Integrated analysis devices and related fabrication methods and analysis techniques

ABSTRACT

Provided are integrated analysis devices having features of macroscale and nanoscale dimensions, and devices that have reduced background signals and that reduce quenching of fluorophores disposed within the devices. Related methods of manufacturing these devices and of using these devices are also provided

RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.16/864,551, filed May 1, 2020, now U.S. Pat. No. 11,292,713, which is acontinuation of U.S. application Ser. No. 15/385,302, filed Dec. 20,2016, now U.S. Pat. No. 10,654,715, which is a continuation of U.S.application Ser. No. 12/996,410, now U.S. Pat. No. 9,533,879, which hasa 371 date of Feb. 16, 2011, and which is the US national phase ofInternational App. No. PCT/US2009/046427, filed Jun. 5, 2009, whichclaims the benefit of U.S. Application No. 61/059,399, filed Jun. 6,2008, each of which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED R&D

This invention was made with government support under Grant No. HG004199awarded by National Institutes of Health. The U.S. Government hascertain rights in this invention.

TECHNICAL FIELD

The present invention relates to the field of nanofluidics and to thefield of solid-state optical analysis devices.

BACKGROUND

One of the challenges in current biomedical analysis is to fully accountfor the complexity of biological samples that may have a great deal ofheterogeneity, and in which samples no two objects are exactly alike.The minority population of cells or molecules in a given sample is oftenthe most clinically relevant portion to the pathophysiological state ofthe patients.

Conventional bulk solution assays can average out and obscure small butsalient features of a heterogeneous sample preventing the earlydiscovery of the disease causal molecules, features and events. Asmolecular biology techniques have evolved, there is increasing interestin analyzing progressively smaller samples with ever-increasingresolution and precision.

The world of single molecule level biology is inherently at the micron-and below scale. One challenge in the field is fabrication of highquality micro- and nanofluidic Structures on solid state materials thatare compatible with existing fabrication processes. The optical purityof the inner surface of a device has a paramount importance innanofluidics designed for single molecule level fluorescent imaging,because optical background contamination generates excessive autofluorescent noise that reduces the effectiveness of the fluidic device.Optical purity, however, is not considered an important aspect inconventional semiconductor fabrication.

An additional challenge facing the field is moving molecules or othertargets from a macroscale environment (e.g., pipettes) to micro- ornano-scale regions, as well as moving such molecules and associatedmedia from the micro- or nano-scale regions to macro-scale waste outletsor sample collection chambers for further downstream analysis.

Such devices must accommodate features having sizes ranging fromcentimeters down to single digit nanometers (a seven orders of magnitudedifference), which represents a tremendously broad range of lengthscales to integrate together in a way that allows for controllable andleak-free transport.

Along with the issues presented by transporting biological and othertargets is the challenge detecting light emitting labels on such targets(e.g., molecules or cellular components of interest), which detectionmay be performed on the target while the target is disposed in anenclosed channel. Such detection has many practical applications,particularly in the field of nanofluidics.

Of particular importance to such detection is the signal-to-backgroundratio (SBR) (also referred to as signal-to-noise ratio, S/N) of thelabel's electromagnetic signal to that of the background signal of thedevice in which the label is contained. Maximizing the SBR by reducingthe background enhances the value of a given system by increasing thedynamic range of that system. The value is further increased by a devicein which the electromagnetic radiation constituting the device'sbackground signal is reduced across the broadest possible spectralrange.

Certain substrates, such as silicon, quench fluorescent emission whenimaging fluorophores on a flat, open silicon substrate, as is commonlydone in microarray-based applications. To prevent this quenching, asubstrate coating is typically employed to reduce or eliminatequenching. However, when incorporated into a bonded fluidic device withconfined channels, the coating material may often increase thebackground signal of the device, which in turn degrades the deviceperformance, and effectively exchanges one problem (quenching) foranother (increased background).

Accordingly, there is a need in the art for devices that exhibit acomparatively low level of background signal while also limiting thequenching of fluorophores or other labels present in the device. Thereis also a need in the art for related methods of fabricating deviceshaving such characteristics.

SUMMARY

In meeting the described challenges, the claimed invention firstprovides analysis devices, comprising a first substrate; a secondsubstrate; a first inlet port extending through at least a portion ofthe first substrate, the second substrate, or both, so as to place afirst interconnector channel in fluid communication with the environmentexterior to the analysis device; and a first front-end branched channelregion, comprising at least a primary channel characterized as having across-sectional dimension in the range of from less than about 10,000 nmand at least two secondary channels, placing the first interconnectorchannel into fluid communication with a nanochannel analysis region, thenanochannel analysis region comprising at least one nanochannelcharacterized as having a cross-sectional dimension less than that ofthe primary channel, and wherein the ratio of the cross-sectionaldimensions of the primary channel to the nanochannel is in the range offrom about 100 to about 10,000.

Also provided are methods of fabricating analysis devices, the methodsincluding bonding a first substrate and a second substrate, at least oneof the substrates comprising at least one channel having a width in therange of from about 10 nm to about 10,000 nm, the bonding giving rise toan enclosed conduit disposed between the substrates, the enclosedconduit being capable of transporting a fluid therethrough.

Further provided are methods of analysis, comprising translocating amacromolecule through at least two channels of successively decreasingwidth such that at least a portion of the macromolecule is elongatedwhile disposed in the narrowest of the channels; the ratio of the widthsof the widest and narrowest channels is in the range of from about 1 toabout 10⁶; detecting a signal from the macromolecule while it resides ina first region of a channel having a width of from 10 nm to about 1000nm; and correlating the signal to a property of the macromolecule.

Further provided are analysis devices, comprising a first substrate anda second substrate, the first and second substrates defining a channeldisposed between the substrates, at least one of the first or secondsubstrates permitting at least partial passage of electromagneticradiation characterized as having at least one wavelength in the rangeof from about 10 nm to about 2500 nm; a first thin film surmounting atleast a portion of the first substrate, the second substrate, or both,at least a portion of the first thin film defining at least a portion ofa channel disposed between the first and second substrates, and thefirst thin film giving rise to a reduced background signal of the devicewhen the device is illuminated by electromagnetic radiation having awavelength in the range of from about 10 nm to about 2500 nm, relativeto an identical device without said first thin film.

Additionally provided are analysis devices, comprising a substrateconfigured so as to define a channel enclosed within the substrate, thesubstrate being transparent to electromagnetic radiation having at leastone frequency component in the range of from about 10 nm to about 2500nm.

Further provided are methods of fabricating an analysis device,comprising disposing a first substrate, a second substrate, and a firstthin film layer so as to define a channel disposed between the first andsecond substrates, the first thin film layer being selected such thatsaid layer reduces the background signal of the device when the deviceis illuminated by electromagnetic radiation having a wavelength in therange of from about 10 nm to about 2500 nm, relative to an identicaldevice without said first thin film; and bonding the first thin filmlayer to the first substrate, the second substrate, or both.

Also provided are methods of fabricating an analysis device, comprisingdisposing a sacrificial template within a workpiece comprising amaterial that is transparent to electromagnetic radiation having awavelength in the range of from about 10 nm to about 5000 nm; removingat least a portion of the sacrificial template so as to give rise to achannel disposed within the workpiece, at least a portion of the channelhaving a cross-sectional dimension in the range of from about 5 nm toabout 5000 nm.

Further provided are methods of analyzing a fluorescently labeledmolecule, comprising placing at least a portion of the fluorescentlylabeled molecule into a channel within an analysis device, the analysisdevice having at least a first substrate, a second substrate, and afirst thin film configured to give rise to the channel being disposedbetween the first and second substrates, the first thin film bonded tothe first substrate, the second substrate, or both, the fluorescentlylabeled molecule capable of emitting electromagnetic radiation of anemission wavelength when the sample is illuminated by electromagneticradiation of an excitation wavelength in the range of from about 10 nmto about 2500 nm, the first thin film reducing the background signal ofthe device when the device is illuminated by electromagnetic radiationof the excitation wavelength, relative to an identical device withoutsaid first thin film, and collecting electromagnetic radiation of theemission wavelength emitted from the fluorescently labeled molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

The summary, as well as the following detailed description, is furtherunderstood when read in conjunction with the appended drawings. For thepurpose of illustrating the invention, there are shown in the drawingsexemplary embodiments of the invention; however, the invention is notlimited to the specific methods, compositions, and devices disclosed. Inaddition, the drawings are not necessarily drawn to scale. In thedrawings:

FIG. 1 depicts a schematic view of a device according to the claimedinvention;

FIGS. 2A-B depict an exemplary device according to the claimedinvention;

FIGS. 3A-B an exemplary fabrication scheme according to the claimedinvention;

FIGS. 4A-B depict an example fabrication scheme for two substrates(substrates A and B; one of the substrates suitably being transparent),with channel elements etched into both substrates;

FIGS. 5A-B depict an exemplary nanodevices having 2 and 4 ports;

FIGS. 6A-B depict an example embodiment of a multi-port device design;

FIGS. 7A-E depict a multi-stage branched channel array according to theclaimed invention;

FIGS. 8A-E illustrate a multi-level, branched, interconnected channelarray;

FIGS. 9A-E illustrate a device design having a combination of branchedchannels and post arrays;

FIGS. 10A-C depict a design having a single long nanochannel arranged ina continuously connected, serial set of parallel nanochannels in aserpentine configuration;

FIGS. 11A-C depict multiple, long nanochannels arranged in acontinuously connected serial set of parallel nanochannels;

FIG. 12 illustrates various, non-limiting embodiments of channel devicesaccording to the claimed invention;

FIG. 13 depicts cross-sectional views of devices according to theclaimed invention, with (a) a channel formed in the lower substrate, (b)channels formed in both the lower and upper substrates, and (c) achannel formed in the upper substrate only—each of these threeembodiments depicts upper and lower thin films;

FIG. 14 depicts cross-sectional views of devices according to theclaimed invention, with (a) a channel formed in the lower substrate, (b)channels formed in the upper and lower substrates, and (c) a channelformed in the upper substrate only—each of these embodiments depictsonly a single thin film that conforms primarily to the lower substrate;

FIG. 15 depicts cross-sectional views of devices according to theclaimed invention, with (a) a channel formed in the lower substrate, (b)channels formed in the upper and lower substrates, and (c) a channelformed in the upper substrate only—each of these embodiments depictsonly a single thin film that conforms primarily to the upper substrate;

FIG. 16 depicts cross-sectional views of devices according to theclaimed invention, with (a) a channel formed in the lower of two thinfilms, (b) channels formed in the upper and lower thin films, and (c) achannel formed in the upper thin film only;

FIG. 17 depicts the operation of a device according to the claimedinvention, showing in (a) the excitation of a fluorescently labeledsample disposed in a device made according to the claimed invention andthe collection of radiation emitted from the excited sample transmittedacross the same substrate and thin film layer across which theexcitation radiation passed, and in (b) the excitation of afluorescently labeled sample disposed in a device made according to theclaimed invention and the collection of radiation emitted from theexcited sample transmitted across a different substrate and thin filmlayer than those across which the excitation radiation passed;

FIG. 18 illustrates background measurements taken at radiationwavelengths of from about 0 nm to about 217 nm of confined channelshaving a SiO_(x) thin film disposed at the bottom of the channel;

FIG. 19 illustrates background measurements taken at radiationwavelengths of from about 0 nm to about 217 nm of confined channelshaving a SiN_(x) thin film disposed at the bottom of the channel;

FIGS. 20A-C illustrate images taken at excitation radiation wavelengthsof about 653 nm of a nanochannel array having a SiO_(x) thin filmdisposed at the bottom of the array and of TOTO-3 labeled DNA residingwithin that array; and

FIGS. 21A-C illustrate images taken at excitation radiation wavelengthsof about 653 nm of a nanochannel array having a SiN_(x) thin filmdisposed at the bottom of the array and of TOTO-3 (fluorophore) labeledDNA residing within that array.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention may be understood more readily by reference to thefollowing detailed description taken in connection with the accompanyingfigures and examples, which form a part of this disclosure. It is to beunderstood that this invention is not limited to the specific devices,methods, applications, conditions or parameters described and/or shownherein, and that the terminology used herein is for the purpose ofdescribing particular embodiments by way of example only and is notintended to be limiting of the claimed invention. Also, as used in thespecification including the appended claims, the singular forms “a,”“an,” and “the” include the plural, and reference to a particularnumerical value includes at least that particular value, unless thecontext clearly dictates otherwise. The term “plurality”, as usedherein, means more than one. When a range of values is expressed,another embodiment includes from the one particular value and/or to theother particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another embodiment. All ranges areinclusive and combinable.

It is to be appreciated that certain features of the invention whichare, for clarity, described herein in the context of separateembodiments, may also be provided in combination in a single embodiment.Conversely, various features of the invention that are, for brevity,described in the context of a single embodiment, may also be providedseparately or in any subcombination. Further, reference to values statedin ranges include each and every value within that range.

Terms

As used herein, “fluidic element” means a feature capable of containingor admitting a fluid, such as a channel, a groove, a trench, anaperture, a portal, a hole, a via, and the like.

As used herein, “cross-sectional dimension” means a width, a diameter, adepth, or other across-wise measurement.

The claimed invention first provides analysis devices. These devicessuitably include, inter alia, a first substrate, and a second substrate.Suitable substrate materials are described elsewhere herein, andinclude, e.g., silicon, glass, and quartz.

The devices also include a first inlet port extending through at least aportion of the first substrate, the second substrate, or both, so as toplace a first interconnector channel in fluid communication with theenvironment exterior to the analysis device.

Also present in the devices is a first front-end branched channelregion, which region includes at least a primary channel characterizedas having a cross-sectional dimension in the range of from less thanabout 10,000 nm and at least two secondary channels, placing the firstinterconnector channel into fluid communication with a nanochannelanalysis region. Branched channel arrangements are shown in, e.g., FIGS.5(b), 7(c) and 8(c), which figures show primary channels divided intosmaller secondary channels.

The nanochannel analysis region suitably includes at least onenanochannel characterized as having a cross-sectional dimension that isless than that of the primary channel. The ratio of the cross-sectionaldimensions of the primary channel to the nanochannel is in the range offrom about 100 to about 10,000, or from about 1000 to about 5000, oreven about 2000.

Substrates may be of many different materials. The first substrate, thesecond substrate, or both is suitably silicon, SiGe, Ge, strainedsilicon, GeSbTe, AlGaAs, AlGaInP, AlGaN, AlGaP, GaAsP, GaAs, GaN, GaP,InAlAs, InAlP, InSb, GaInAlAs, GainAlN, GaInAsN, GaInAsP, GalnAs, GaInN,GaInP, GaSb, InN, InP, CdSe, or CdTe. Zinc compounds, such as zincselenide (ZnSe), HgCdTe, ZnO, ZnTe, and zinc sulfide (ZnS) are alluseful.

A listing of substrate materials also includes aluminum, aluminum oxide,stainless steel, Kapton™, metal, ceramic, plastic, polymer, sapphire,silicon carbide, silicon on insulator (SOI), astrosital, barium borate,barium fluoride, sillenite crystals BGO/BSO/BTO, bismuth germanate,calcite, calcium fluoride, cesium iodide, Fe:LiNbO₃, fused quartz,quartz, fused silica, glass, SiO₂, gallium, gadolinium garnet, potassiumdihydrogen phosphate (KDP), thalium bromoiodide (KRS-5), potassiumtitanyl phosphate, lead molibdate, lithium fluoride, lithium iodate,lithium niobate, lithium tantalate, magnesium fluoride, potassiumbromide, titanium dioixde, sodium chloride, tellurium dioxide, zincselenide, spin-on glass, UV curable materials, soda lime glass, anycompound above in hydrogenated form, stoichiometric variations of theabove compounds, or any combinations thereof. In some embodiments, asubstrate is optically opaque, in others, a substrate is essentiallytransparent to visible light or to at least one wavelength ofelectromagnetic radiation.

The first substrate suitable has a thickness in the range of from about10 nm to about 10,000 nm, or from about 100 nm to about 1000 nm, or fromabout 200 nm to about 500 nm. The second substrate may have a thicknessin the same range; the two substrates may of the same thickness or ofdifferent thicknesses.

An inlet port is suitably circular in cross-section (e.g., FIG. 1 ),although other profiles may be used. An inlet port suitably has adiameter or other cross-sectional dimension in the range of from about 5microns to about 5000 microns, or from about 10 microns to about 100microns, or about 50 microns. The inlet port may extent through thethickness of a substrate, or partially through the substrate. The portmay be plugged or capped, and can also include a valve or other seal.

Outlet ports suitably have dimensions similar to those of inlet ports,although inlet and outlet ports on a given device need not be of thesame dimensions. A port suitably extends through the entire thickness ofa substrate, although inlets (and outlets) that extend through only aportion of a substrate may also be used.

An interconnector channel of the claimed invention suitably has a depthin the range of from about 100 nm to about 100 microns, or from about500 nm to about 50 microns, or from about 1 micron to about 10 microns.The interconnector also suitably has a width in the range of from about500 nm to about 1000 microns, or from about 1 micron to about 50microns, or from about 10 microns to about 50 microns. Interconnectregions are shown in, e.g., FIG. 5 .

An interconnector may in some configurations connect two—or more—inlets,and may also be in fluid communication with one, two, three, or moreprimary channels of branched regions, as shown in FIG. 5 . In someembodiments, the branched region is in direct fluid communication withthe inlet port, without an intervening interconnector region.

In the branched (or furcated) regions of the claimed devices, theprimary channel suitably has a width in the range of from about 10 nm toabout 10,000 nm, or in the range of from about 50 nm to about 1000 nm,or in the range of from 75 nm to about 200 nm. The optimal with of aprimary channel will depend on the needs of the user.

Primary channels can have a depth in the range of from about 10 nm toabout 1000 nm, or from about 50 nm to about 500 nm, or even from about100 nm to about 200 nm.

The front-end branched channel region suitably includes a splitterstructure that divides the primary channel into least two secondarychannels, as shown in, e.g., FIG. 7 . In some embodiments (see FIG. 7 ),the splitter structure includes at least one surface angled in the rangeof from about 0 and about 90 degrees relative to the centerline of theprimary channel. In the non-limiting embodiment shown in FIG. 7 , thesplitter includes a surface angled between 0 and 90 degrees relative tothe centerline of the primary channel shown at the top of FIG. 7(c).

The width of a secondary channel in such embodiments is suitably in therange of from about 30% to about 70% of the width of the primarychannel, or about 45% to 55% of the primary channel. In someembodiments, the cross-sectional area of a secondary channel is about50% of the cross-sectional area of the primary channel. In otherembodiments, one of the secondary channels differs in cross-sectionalarea, width, depth, or some combination thereof from the other secondarychannel. In other embodiments, the secondary channels are of similar oreven identical dimensions to each other.

A secondary channel may have a length in the range of from about 1microns and about 500 microns, or from about 10 microns to about 100microns. Secondary channels may have the same or different lengths.

In some embodiments (e.g., FIG. 7 , FIG. 8 ), a secondary channel isdivided into two tertiary channels by a splitter having at least onesurface angled in the range of from about 0 and about 90 degreesrelative to the centerline of the secondary channel. This is shown bythe non-limiting embodiment of FIG. 7 .

In some configurations of the claimed invention, the splitter structureincludes a contoured portion, such as that shown in FIG. 8 . Suchsplitter structures are suitably configured such that a fluid borne bodypropelled through the primary channel by a gradient is essentiallyequally likely to enter either secondary channel downstream from thesplitter structure, as shown in FIG. 8(c). As shown in that figure, thesplitter is shaped and configured such that field lines of an electricfield applied across the device will result in targets (e.g., DNA orother biopolymers) that pass through the region being distributedessentially equally across the four tertiary channels shown at thebottom of the figure.

The splitter may be configured so as to define an overhang that shieldsat least a portion of the secondary channel from the primary channel, asshown in FIG. 8 . The overhang may be configured such that the overhangis in the range of from about 5% to about 50% of the width of thesecondary channel.

The width of a secondary channel may be in the range of from about 30%to about 70% of the width of the primary channel, or even 50% of theprimary channel. As described elsewhere herein, a secondary channel mayhave a cross-sectional area that is in the range of from about 30% to70% of the cross-sectional area of the primary channel, or even about50% of the cross-sectional area of the primary channel.

A nanochannel in the nanochannel analysis region of the claimed devicessuitably has a width in the range of from about 1 nm to about 1000 nm,or from about 10 nm to about 100 nm, or even from about 50 nm to about80 nm. The nanochannel can have a depth in the range of from about 10 nmto about 500 nm, or from about 20 nm to about 200 nm, or even from about50 nm to about 100 nm.

In some configurations, the nanochannel has at least one linear segmenthaving a length in the range of from about 0.1 microns to about 50microns. Linear segments are shown in FIGS. 10A-C, FIG. 11 , and FIG. 12. The nanochannel may include a bend or curve of at least about 30degrees, at least about 90 degrees, or even a bend of about 180 degreesor more. In some embodiments, a nanochannel is circular or can even bein a spiral configuration.

A nanochannel may possess a constant width and depth, but may also havewidth that varies, a depth that varies, or both. Channels may be zig-zagin form (FIG. 12 ), or may have an undulating floor, giving the channela varying depth along its length.

In some embodiments, like that shown in FIG. 5(b), the nanochannelanalysis region is in fluid communication with a first back-end branchedchannel region. Back-end branched regions are suitably similar to thepreviously described front-end branched regions, and can becharacterized as being downstream from the front-end branched channelregion. The front- and back-end regions on a given device may be thesame or differ from one another. The devices may also include a secondinterconnector channel (FIG. 5(b)) that is in fluid communication with aport (inlet or outlet), with a branched region (FIG. 5(b)), or both. Aprimary channel may also be in fluid communication with a secondinterconnector channel, or even with a second (e.g., outlet) port.

In some embodiments, the ratio of a cross-sectional dimension of theport to a cross-sectional dimension of the at least one nanochannel isin the range of from about 1 to about 10⁷. In some cases, the ratio is100, 1000, or even 10,000. The ratio demonstrates that the claimeddevices are suitable for transporting (and also analyzing) a target thatis transported from a micro- (or larger) scale environment to anano-scale environment.

This ability to controllably translocate targets from a macroscaleenvironment to a micro- or nano-scale environment is of great valuebecause it enables a user to begin with a large volume of sample(typically molecules or other targets dispersed in a fluid) and thenutilize devices according to the claimed invention to controllablyisolate a single targets from that large sample. Moreover, the claimedinventions allow the user to isolate that single target in a nanoscaleenvironment, such as a channel. The claimed invention thus enables auser to perform single-molecule analysis on an individual molecule thatis formerly dispersed—with many other molecules—in a large volume ofmedia.

In some embodiments, the nanochannel analysis region and a branchedchannel region are disposed in the same plane. In others, they are indifferent planes. The nanochannel analysis region can be is in fluidcommunication with a second nanochannel analysis region, the secondnanochannel analysis region being disposed in a different substrate thanthe first nanochannel analysis region. In such embodiments, stacked orthree-dimensional multi-analysis region devices may be constructed, andmeta-devices that include multiple nanochannel analysis regions may beconstructed.

Also provided are methods of fabricating analysis devices. These methodsinclude, inter alia, bonding a first substrate and a second substrate,at least one of the substrates including at least one channel having awidth in the range of from about 10 nm to about 10,000 nm, the bondinggiving rise to an enclosed conduit disposed between the substrates, theenclosed conduit being capable of transporting a fluid therethrough.

Bonding may be accomplished by anodic bonding, thermal bonding, or anycombination thereof. Chemical bonding may also be used. Sample processconditions for anodic bonding of a Si-glass device are describedelsewhere herein.

The methods can include disposing a thin film atop at least a portion ofthe first substrate, the second substrate, or both, which thin film maybe disposed within at least a portion of any channels disposed in thesubstrate. The film may be used to enhance bonding between thesubstrates.

As one non-limiting example, a silicon dioxide (or silicon nitride) filmmay be used to enhance (or even enable) bonding between a siliconsubstrate and a glass or other substrate. The thin film may also bechosen so as to electrically insulate at least a portion of the interiorof the enclosed conduit from at least one of the substrates. Asdescribed elsewhere herein in more detail, a thin film may be used toshield at least a portion of the conduit from a substrate, which canprevent the substrate from quenching a fluorophore disposed within theconduit.

The thin film may be disposed so as to reduce the cross-sectional areaof the enclosed conduit to a predetermined value, which reduction isaccomplished by building up the floor and sides of a channel so as toreduce the cross-sectional area available to a fluid flowing within theconduit. The thin film may be disposed to reduce the cross-sectionalarea by at least about 1%, at least about 5%, or even by at least about10% or even 25%. The thin film can even be disposed to completely fillthe channel. Channels can be etched in the film, as shown in, e.g., FIG.4 and FIG. 16 .

A substrate can include two or more channels. Two of the substrates mayeach include at least one channel such that the bonding gives rise totwo or more enclosed conduits disposed between the substrates. Inembodiments where both substrates include a channel, the substrates maybe bonded so that the channels are in at least partial registration withone another (e.g., FIG. 13 ).

In some embodiments, the ratio of the widths of two conduits of theresultant device is in the range of from about 1 to about 10⁷, or in therange of from about 100 to about 10,000, or is even about 1000.

In some embodiments, the first substrate, the second substrate, or both,includes a dielectric. The first substrate, the second substrate, orboth, can include a semiconducting material, or even a conductingmaterial. One or both of the substrates is suitably transparent to atleast one wavelength of electromagnetic radiation, or even transparentto visible light.

Also provided are methods of analysis. The methods suitably includetranslocating a macromolecule through at least two channels ofsuccessively decreasing width such that at least a portion of themacromolecule is elongated while disposed in the narrowest of thechannels, the ratio of the widths of the widest and narrowest channelsbeing in the range of from about 1 to about 10⁷, or even from about 100to about 10⁵. In some embodiments, the macromolecule is translocatedthrough a single channel of decreasing width or cross-sectional area,various widths along the channel being in accordance with theaforementioned ratio.

In some embodiments, the user may translocate a target through an inlethaving a cross-sectional dimension in the centimeter range, with thetarget ultimately arriving at a channel having a cross-sectionaldimension in the nanometer range.

The methods also include detecting a signal from the molecule while itresides in a first region of a channel having a width of from 10 nm toabout 1000 nm, or from about 50 nm to about 500 nm, or from about 100 nmto about 200 nm.

The user can then correlate the signal to a property of themacromolecule. For example, after exposing the sample to a fluorescenttag that binds to a unique DNA sequence on a sample, the user can theninterrogate the sample to determine whether the fluorescent tag ispresent (or not present) on the sample. The user may also correlate theduration of the signal to the length or other property of themacromolecule, or even the macromolecule's velocity through the device.

The signal need not be emitted by a fluorescent molecule; the signal canbe magnetic or radioactive. In some embodiments, the user may opticallyinspect the target while it is disposed in a channel (or conduit). Thesignal may be a signal evolved from exciting a label, or it may be asignal or reflection that is effected by illuminating the sample. Inembodiments where optical inspection of the sample may be performed, orwhere the signal includes electromagnetic radiation, it isdesirable—though not necessary—for at least one of the substrates (andany intervening thin films) to be transparent.

Translocation may be accomplished by application of an electricalgradient, a pressure gradient, a magnetic field, a thermal gradient, orany combination thereof. The translocation may include applying aconstant gradient, or a varying gradient.

The methods further include translocating the macromolecule through atleast two channels of successively increasing width. In someembodiments, the direction of the gradient may be reversed so as toreverse the direction of the macromolecule such that at least a portionof the macromolecule re-enters the first region of the channel. The usercan thus move a target macromolecule back and forth within a givendevice.

This back-and-forth control, akin to advancing and rewinding a tape in atape player, is useful in analyzing a macromolecule or other targetbecause the user may pass the target through the nanochannel analysisregion and then “rewind” the macromolecule by reversing the gradient,and then re-analyze the same molecule. This enables the user to easilyrepeat measurements of a given target, allowing the user to quicklyassemble a large (i.e., statistically useful) set of measurements. Theability to adjust the gradient also allows a user to quickly advance (or“fast forward”) a target through one portion of the analysis device, andthen slow the target down for analysis.

Detection is suitably accomplished optically, electrically,magnetically, electromagnetically, or combinations thereof. Photoncounters and microscopes are suitable for performing detection accordingto the claimed methods.

In another aspect, the present invention provides analysis devices.These devices suitably include a first substrate and a second substrate,the first and second substrates defining a channel disposed between thesubstrates, at least one of the first or second substrates permitting atleast partial passage of electromagnetic radiation characterized ashaving at least one wavelength in the range of from about 10 nm to about2500 nm; a first thin film surmounting at least a portion of the firstsubstrate, the second substrate, or both.

The thin film can be a single layer of material. A substrate may besurmounted by multiple films, and a thin film may itself be composed ofa single material or a combination of materials. A substrate may besurmounted by one, two, three, or more discrete thin films. In someembodiments, the substrate or thin film may act as a waveguide orillumination source, so as to enhance observation of a target disposedwithin the device.

At least a portion of the first thin film suitably defines at least aportion of a channel disposed between the first and second substrates,the first thin film giving rise to a reduced background signal of thedevice when the device is illuminated by electromagnetic radiationhaving a wavelength in the range of from about 10 nm to about 2500 nm,relative to an identical device without said first thin film.

The thin film is suitably bonded to the first substrate, the secondsubstrate, or both. The substrates are suitably bonded to one another,and the bonding may be through the thin film or thin films. In someembodiments, a thin film is bonded to a substrate. Thin films may, insome embodiments, be bonded to one another.

The first thin film suitably includes silicon nitride. The first thinfilm may also include, e.g., silicon oxynitride, SiO_(x)N_(y),hydrogenated silicon dioxide, hydrogenated silicon nitride, hydrogenatedsilicon oxynitride, high K dielectrics, compounds including titanium:TiSiO, TiO, TiN, titanium oxides, hydrogenated titanium oxides, titaniumnitrides, hydrogenated titanium nitrides, TaO, TaSiO, TaOxNy, Ta₂Os,TaCN, tantalum oxides, hydrogenated tantalum oxides, tantalum nitrides,hydrogenated tantalum nitrides.

Compounds that include hafnium are also suitable, and include HfO₂,HfSiO₂, HfZrO_(x), HfN, HfON, HfSiN, HfSiON, hafnium oxides,hydrogenated hafnium oxides, hafnium nitrides, hydrogenated hafniumnitrides, ZrO₂, ZrSiO₂, ZrN, ZrSiN, ZrON, ZrSiON, zirconium oxides,hydrogenated zirconium oxides, zirconium nitrides, hydrogenatedzirconium nitrides, Al₂O₃, AlN, TiAlN, TaAlN, WAlN, aluminum oxides,hydrogenated aluminum oxides, aluminum nitrides, hydrogenated aluminumnitrides.

Suitable materials also include WN, low K dielectrics, fluorine dopedsilicon dioxide, carbon doped silicon dioxide, porous silicon dioxide,porous carbon doped silicon dioxide, spin-on organic polymericdielectrics, graphite, graphene, carbon nano-tubes, plastics, polymer,organic molecules, self-assembled monolayers, self-assembledmulti-layers, a lipid bi-layer, any of the aforementioned compounds inan hydrogenated form, a stoichiometric variation of any of theforegoing, and combinations thereof.

The first substrate, the second substrate, or both, may include glass,silicon, or a combination of the two. In some embodiments, one or bothof the substrates includes quartz, fused silica, sapphire, siliconcarbide, soda lime, germanium, silicon germanium, gallium, indium,cadmium, zinc, aluminum, stainless steel, Kapton™ polymeric material, apolymer, a semiconductor material, a metal, a ceramic, and the like. Thesubstrates may also include combinations of these materials.

At least one of the substrates is suitably transparent to at least onefrequency of electromagnetic radiation. In some embodiments, one or bothof the substrates is essentially transparent to visible light. Thistransparency facilitates the observation of targets (e.g., fluorescentlylabeled macromolecules) that may be disposed within the devices.

Suitable glasses include Schott Borofloat™ 33 glass, Pyrex 7740™ glass,Hoya SD2™ glass, combinations thereof, and the like.

Substrates suitably have a thickness in the range of from about 0.01 mmto about 5 mm, or from about 0.1 mm to about 1 mm, or even about 0.5 mm.

The first thin film may have a thickness in the range of from about 1 nmto about 5000 nm, or from about 10 nm to about 1000 nm, or from about 50nm to about 500 nm, or even from about 100 nm to about 200 nm.

The conduits of the claimed devices suitably have a width in the rangeof from about from about 5 nm to about 5 mm, or from about 10 nm toabout 1 mm, or from 50 nm to about 1 micron, or from about 100 nm toabout 500 nm. The channels suitably have a depth in the range of fromabout 5 nm to about 1 mm, or from about 100 nm to about 1000 nm.

The devices may also include a second thin film. The second thin film issuitably chosen so as to give rise to a reduced background signal of thedevice when the device is illuminated by electromagnetic radiationhaving a wavelength in the range of from about 10 nm to about 2500 nm,relative to an identical device without said second thin film. Siliconnitride is considered especially suitable for use as a thin film.

Other materials may also be used in the second thin film. Thesematerials include, inter alia, silicon oxynitride, SiOxNy, hydrogenatedsilicon dioxide, hydrogenated silicon nitride, hydrogenated siliconoxinitride, high K dielectrics, compounds including titanium: TiSiO,TiO, TiN, titanium oxides, hydrogenated titanium oxides, titaniumnitrides, hydrogenated titanium nitrides, TaO, TaSiO, TaOxNy, Ta₂O₅,TaCN, tantalum oxides, hydrogenated tantalum oxides, tantalum nitrides,hydrogenated tantalum nitrides, compounds containing hafnium: HfO₂,HfSiO₂, HfZrO_(x), HfN, HfON, HfSiN, HfSiON, hafnium oxides,hydrogenated hafnium oxides, hafnium nitrides, hydrogenated hafniumnitrides, ZrO₂, ZrSiO₂, ZrN, ZrSiN, ZrON, ZrSiON, zirconium oxides,hydrogenated zirconium oxides, zirconium nitrides, hydrogenatedzirconium nitrides, Al₂O₃, AlN, TiAlN, TaAlN, WAlN, aluminum oxides,hydrogenated aluminum oxides, aluminum nitrides, hydrogenated aluminumnitrides, SiN, WN, low K dielectrics, fluorine doped silicon dioxide,carbon doped silicon dioxide, porous silicon dioxide, porous carbondoped silicon dioxide, spin-on organic polymeric dielectrics, graphite,graphene, carbon nano-tubes, plastics, polymer, organic molecules,self-assembled monolayers, self-assembled multi-layers, a lipidbi-layer, any of the aforementioned compounds in an hydrogenated form, astoichiometric variation of any of the foregoing, combinations thereof,and the like.

The second thin film suitably has a thickness in the range of from about1 nm to about 5000 nm, or from about 100 nm to about 1000 nm, or evenfrom about 300 nm to about 500 nm. A thin film may be selected so as toprevent or reduce quenching of a fluorescent molecule disposed withinthe device by exposure to the first substrate, second substrate, orboth. A thin film may also be selected so as to reduce the backgroundsignal evolved from the device.

The present invention also provides analysis devices. These devicessuitably include a substrate configured so as to define a channelenclosed within the substrate, and the substrate being transparent toelectromagnetic radiation having at least one frequency component in therange of from about 10 nm to about 2500 nm.

The channel is suitably characterized as being a conduit, although otherconfigurations are within the scope of the invention. The channel alsosuitably has at least one cross-sectional dimension (e.g., width,diameter) in the range of from about 5 nm to about 5 mm, or in the rangeof from about 50 nm to about 500 nm, or even about 75 nm to about 100nm. The channel is suitably formed in silicon nitride, although othermaterials that are essentially transparent to at least one wavelength ofelectromagnetic radiation may be used.

Silicon nitride is considered especially suitable because, as describedelsewhere herein, the material is sufficiently transparent to visiblelight (and other wavelengths) to facilitate observation of a sampledisposed within. Further, silicon nitride—as shown in FIG. 19 —does noteffect quenching of flurophores disposed nearby, which furtherfacilitates analysis of labeled targets disposed within the devices.

Also provided are methods of fabricating analysis devices. These methodsinclude, inter alia, disposing a first substrate, a second substrate,and a first thin film layer so as to define a channel disposed betweenthe first and second substrates.

The first layer is suitably selected such that the layer reduces thebackground signal of the device when the device is illuminated byelectromagnetic radiation having a wavelength in the range of from about10 nm to about 2500 nm, relative to an identical device without the thinfilm. The first thin film layer is suitably bonded to the firstsubstrate, the second substrate, or both.

Some substrates (e.g., quartz to quartz) may be bonded directly to oneanother. In some embodiments, the substrates are bonded to one anotherthrough a thin film; a thin film may be bonded to one or moresubstrates, and may even be bonded to another thin film. As describedelsewhere, a thin film (e.g., an oxide) can enhance (or even enable)bonding between two substrates.

A second thin film layer may be bonded to the first substrate, thesecond substrate, the first thin film layer, or combinations thereof.Bonding may be anodic, thermal, chemical, or by other methods known tothose of skill in the art.

The first thin film layer (or other thin film layers) are suitablyselected such that the thin film layer reduces (or otherwise minimizes)quenching of fluorophores disposed within the device. Without beingbound to any particular theory, the thin film may act as a shieldbetween the fluorophore and one or more of the device's substrates.

In some embodiments, the thin film serves to provide physical separationbetween the fluorophore and the substrate; without the thin film, thefluorophore would reside relatively close to the substrate material, andthe fluorophore's may be reduced or otherwise quenched by the substratematerial as the fluorphore resides in a channel that acts as a “darkwell.” Silicon nitride is considered a suitable material for reducingquenching.

Also provided are methods of fabricating analysis devices. These methodsinclude disposing a sacrificial material or template within a workpieceincluding a material that is transparent to electromagnetic radiationhaving a wavelength in the range of from about 10 nm to about 5000 nm.The user then removes at least a portion of the sacrificial template soas to give rise to a channel disposed within the workpiece, and at leasta portion of the channel having a cross-sectional dimension in the rangeof from about 5 nm to about 5000 nm.

In one embodiment, a tube, cord, or other sacrificial material isembedded in the radiation-transparent material; this may be accomplishedby lithographic processes, by softening the radiation-transparentmaterial, or by other methods. The sacrificial material is thenremoved—by heating, etching, vaporizing, or other process—so as to leavebehind a channel in the radiation-transparent substrate. Controlling thedimensions and orientation of the sacrificial material thus enables theuser to achieve channels of various size and geometry.

The channels suitably have at least one cross-sectional dimension (e.g.,diameter, width, or even depth) in the range of from about 5 nm to about5000 nm, or from about 10 nm to about 1000 nm, or from about 50 nm toabout 500 nm. The channel may have a constant cross-section or a varyingcross-section. A given device may include two or more channels, whichchannels may be in fluid communication with one another.

Also provided are methods of analyzing fluorescently labeled molecules.The methods include placing at least a portion of the fluorescentlylabeled molecule into a channel within an analysis device, the devicesuitably having at least a first substrate, a second substrate, and afirst thin film configured to give rise to the channel being disposedbetween the first and second substrates.

The devices suitably include a first thin film bonded to the firstsubstrate, the second substrate, or both. The fluorescently labeledmolecule is suitably capable of emitting electromagnetic radiation of anemission wavelength when the sample is illuminated by electromagneticradiation of an excitation wavelength in the range of from about 10 nmto about 2500 nm, and the first thin film suitably reduces thebackground signal of the device when the device is illuminated byelectromagnetic radiation of the excitation wavelength, relative to anidentical device without said first thin film. The user then collectselectromagnetic radiation of the emission wavelength emitted from thefluorescently labeled molecule.

The background signal of the device is attributable to the firstsubstrate, the second substrate, or both. The addition of a thin filmcan, in some embodiments, increase the background signal of the device(e.g., silicon dioxide).

Devices according to the claimed invention may include two substrates,with one or more channels etched into the base substrate, thetransparent substrate, or both, as shown in non-limiting FIG. 13 . Asshown in that figure, the base substrate is given a “bottom thin film”before bonding to reduce the background, and the transparent substrate(in some embodiments) can also be given a “top thin film”.

The bottom and top thin films suitably conform to the transparent andbase substrates, also as shown in FIGS. 13(a), (b), and (c). One or moreof the thin films is suitably bound to one or more of the substrates. Insome embodiments, thin films may be bonded to one another, andsubstrates may also be bonded to one another. In some embodiments,channels are formed in facing substrates, coatings, or both, and thechannels may be placed in registration with one another so as to giverise to a “combined” channel that is defined by two channels (FIG.13(b), FIG. 14(b), FIG. 15(b), and FIG. 16(b), for example) placed intoregistration with one another.

A substrate, or a thin film, may have channels, pillars, ramps, bumps,or even notches formed thereon. In some embodiments, substrates bondedto each other each have different features patterned and etched thereonsuch that bonding the substrates to one another results in a devicehaving a combination of the substrates' features. As one non-limitingexample, an upper substrate may be etched with a set of comparativelywide channels, and a lower substrate may be patterned with an array ofmicropillars, positioned such that when the substrates are bondedtogether, the pillars of the lower substrate are disposed within thechannels of the upper substrate. Such a device might be similar to thedevices shown in FIG. 9 .

In some embodiments, one or more valves are used to modulate fluid flowwithin a device. As one example, a valve may be disposed at the inlet oroutlet of a device.

FIG. 14 and FIG. 15 depict devices having two substrates and only asingle thin film layer. The single thin film layer suitably conforms toat least one of the substrates, as shown in FIG. 14 (bottom thin film onbase/lower substrate) and FIG. 15 (top thin film on upper, transparentsubstrate). There may also (not shown) be embodiments having a singlesubstrate and a single thin film, the channel being defined by only thatsingle substrate and that single thin film.

FIG. 16 and FIG. 17 illustrate additional embodiments. As shown in thosefigures, a channel may be formed in a thin film (as contrasted with in asubstrate, as shown in FIG. 13 , FIG. 14 , and FIG. 15 ). In thesefurther configurations, planar substrates may be used, and the thin filmmay be disposed (e.g., deposited, grown, etc.) so as to give rise to atrench, slot, or other channel. Alternatively, the thin film may bedisposed, followed by removal of part of the thin film (e.g., byetching, ablation, or by other techniques) so as to give rise to achannel of the desired dimensions and orientation.

In other embodiments (FIG. 14(b)), channels may be formed in both asubstrate and a thin film layer, depending on the user's needs. Thechannels or channels may be formed in a thin film on the upper or lowersubstrate.

The confined channel suitably contains, during operation, a medium inwhich labeled bodies of interest (e.g., FIG. 17 ). Suitably, the labeledbodies include fluorophores that are fluorescently excited in thechannels by passing electromagnetic radiation through a transparentsubstrate (and, in some embodiments, a thin film), with the excitedlabels then emitting an electromagnetic radiation signal back through atransparent substrate, where the emission is then detected (FIG. 17(a)).

Other potential embodiments include those configurations that usemultiple energy transfer steps (such as fluorescence resonance energytransfer, “FRET”) before the electromagnetic radiation signal is emittedfrom the confined channel, through the transparent substrate. FIG. 17 isexemplary only, and other detection schemes may be used in connectionwith the claimed invention; FIG. 17(b) shows an embodiment where thebase substrate is transparent to the wavelength of the signal'selectromagnetic radiation. The user may also detect a magnetic,radioactive, or electrical signal.

Transparent Layer

The transparent substrate (e.g., the upper substrate in FIG. 13 ) issuitably a material capable of being permanently bonded to the basesubstrate, or is transparent to the electromagnetic radiation in thefrequency of interest, or both.

Suitable substrate material is a glass or other material that permits atleast partial passage of visible light, while also having similarthermal expansion characteristics to that of the base substrate in thetemperature range of about 0° C. to about T_(b), where T_(b) is thebonding temperature. The glass may suitably be Schott Borofloat 33™,Pyrex 7740™, or Hoya SD2™, and base substrate silicon.

Other suitable substrates include quartz, fused silica, glass, fusedquartz, sapphire, silicon carbide, and soda lime glass. The substratethickness is suitably between 0.01 mm to 5 mm, or even between 0.01 and0.3 mm. The substrate may be of uniform thickness or of varyingthickness.

The device can be in the form of a chip, slide, or other insertableform. The devices may be inserted into a reader/detector device, or thedevice may be incorporated into a reader/detector device. The device mayinclude one or more chambers or channels for performing analysis, whichanalysis may be performed on multiple samples in parallel.

The bonding process is suitably any process that can permanently bondthe transparent and base substrates, such as anodic bonding. Otherbonding processes include, but are not limited to: fusion, thermal,direct, plasma-activated, chemically-activated, dielectric polymer, andadhesive bonding schemes.

Bottom Thin Film

The bottom thin film (e.g., shown in FIG. 13 ) is suitably of adifferent composition from the base substrate, and acts to reduce thebackground signal of the channel and the surrounding region. This thinfilm material can be applied by growth, deposition, evaporation,sputtering, spin-thin film, lamination, or plating onto the basesubstrate. The material can be applied after the etching of the channelsor other fluidic elements, or before channels or other structures areetched, in which case the channels or other structures (e.g., fluidicelements) are etched into the thin film, as shown in FIG. 16 ).

The material can be thermally grown if it is silicon dioxide, ordeposited by a low-pressure chemical vapor deposition (LPCVD) or atomiclayer deposition (ALD) process, where the material is silicon nitride.

A variety of deposition/application methods may be used for the bottomthin film, including: Physical vapor deposition (PVD), chemical vapordeposition (CVD), plasma enhanced chemical vapor deposition (PECVD),atmosphere pressure CVD (APCVD), ultrahigh vacuum CVD (UHVCVD), aerosolassisted CVD (SSCVD), Direct liquid injection CVD (DLICVD), microwaveplasma assisted CVD (MPCVD), atomic layer deposition (ALD), atomic layerCVD, epitaxy, molecular beam epitaxy (MBE), metalorganic vapor phaseepitaxy (MOVPE), organometallic vapor phase epitaxy (OMVPE),metalorganic chemical vapor deposition (MOVCD), organometallic chemicalvapor deposition (OMCVD), Vapor phase epitaxy (VPE), plating,evaporation, thermal evaporation, electron beam evaporation, pulsedlaser deposition, cathodic arc deposition, sputtering, chemical solutiondeposition, spin thin film, langmuir blodgett film, spray thin film, andthe like.

The bottom thin film material thickness can vary from about 1 nm toabout 5000 nm, or from about 500 nm to about 1000 nm. The thickness neednot be uniform, and is suitably between about 20 and about 500 nm. Asshown in the attached figures, the thin film may conform to the surfaceprofile of the substrate that the thin film contacts.

The thin film material is suitably a material that is at least partlyelectrically insulating. The material selection can be silicon nitride(SiN_(x) or SisN₄). Other possibilities include, but are not limited to:dielectrics, ceramics, silicon dioxide (SiO₂), silicon oxide, glass,quartz, fused silica, SiO_(x), silicon oxinitride, SiN_(x)O_(y),hydrogenated silicon dioxide, hydrogenated silicon nitride, hydrogenatedsilicon oxinitride.

High K dielectrics and compounds containing titanium (TiSiO, TiO, TiN,titanium oxides, hydrogenated titanium oxides, titanium nitrides,hydrogenated titanium nitrides) are also suitable. Similarly, compoundscontaining tantalum: TaO, TaSiO, TaO_(x)N_(y), Ta₂Os, TaCN, tantalumoxides, hydrogenated tantalum oxides, tantalum nitrides, hydrogenatedtantalum nitrides are suitable.

Hafnium compounds, such as HfO₂, HfSiO₂, HfZrO_(x), HfN, HfON, HfSiN,HfSiON, hafnium oxides, hydrogenated hafnium oxides, hafnium nitrides,hydrogenated hafnium nitrides, zirconium compounds (ZrO₂, ZrSiO₂, ZrN,ZrSiN, ZrON, ZrSiON, zirconium oxides, hydrogenated zirconium oxides,zirconium nitrides, hydrogenated zirconium nitrides are also suitable.Aluminum compounds, including Al₂O₃, AlN, TiAlN, TaAlN, WAlN, aluminumoxides, hydrogenated aluminum oxides, aluminum nitrides, andhydrogenated aluminum nitrides are useful.

SiN, WN, Low-K dielectrics, fluorine doped silicon dioxide, carbon dopedsilicon dioxide, porous silicon dioxide, and porous carbon doped silicondioxide are also suitable. Some embodiments may include spin-on organicpolymeric dielectrics, graphite, graphene, carbon nano-tubes, plastics,polymer, organic molecules, self-assembled monolayers, self-assembledmulti-layers, lipid bi-layers, or any of the aforementioned compounds inan hydrogenated form, stoichiometric variations of the above compounds(e.g., SiO_(x) rather than SiO₂; Ta_(x)O_(y) instead of Ta₂Os),combinations thereof, and the like.

The bottom thin film material, application, morphology, and topology aresuitably chosen such that it reduces the effective background signal ofthe device relative to the signal evolved from a body of interestdisposed within the channel, and suitably also reduces or even minimizesthe quenching of fluorescent (or other) labels used to observe thesamples being analyzed. With this guideline in mind, those of ordinaryskill will encounter little difficulty in selecting the optimal thinfilm in view of the signal evolved from the channel at the one or morewavelengths being used to evaluate (i.e., excite) the body of interest,and, in some embodiments, to optimize the signal-to-background levels.

Top Thin Film

The top thin film material's composition, application procedure,topology, morphology and thickness range are suitably the same as thebottom thin film, except that the top thin film is applied to the uppertransparent substrate instead of the lower substrate, and that it maynot necessarily be present in a particular chip embodiment.

The top or upper thin film material, application, morphology, andtopology are suitably chosen so as to reduce the effective backgroundsignal of the device relative to the signal evolved from a body ofinterest disposed within the channel, and suitably also reduces or evenminimizes the quenching of fluorescent (or other) labels used to observethe samples being analyzed. With this guideline in mind, those ofordinary skill will encounter little difficulty in selecting the optimalthin film in view of the signal evolved from the channel at the one ormore wavelengths being used to evaluate (i.e., excite) the body ofinterest, and, in some embodiments, to optimize the signal-to-backgroundlevels.

Confined Channel

The confined channel's width can vary from about 5 nm to about 5 mmwithin the channel. The confined channel depth suitably varies fromabout 5 nm to about 1 mm within the channel. The confined channel widthcan vary from about 5 nm to about 50 microns within the channel, and theconfined channel depth of from about 5 nm to about 50 microns within thechannel. In some embodiments, the channel defines a channel of uniformdepth and cross-section, although a channel may have a varying depth orcross-section as may be dictated by the needs of the user. As oneexample, a channel may narrow from a comparatively wide inlet down to anarrower passage or channel, or may broaden from a narrow inlet. Thechannel may, as shown in the attached figures, include various obstaclesor other structures that extend from the channel's floor to its ceiling,or extend along at least part of the channel's height, as shown in FIGS.20A-C and FIGS. 21A-C, which figures show (looking downward) the tops ofobstacles that are channel or rectangular in cross-section. Obstaclesmay be pillars, curves, and the like.

The confined channels suitably contain the bodies of interest in amedium, which medium can be a fluid, e.g., a liquid. Suitable mediainclude gas, liquid, solids, plasma, vacuum, vapor, colloids,combinations thereof, and the like. The medium can be a buffer, apreservative, and the like.

Channels can be singular or multiple, and two or more channels may beconnected to one another and, in some embodiments, may be connected to acommon reservoir. The channels may be arrayed or multiplexed so as toallow for simultaneous analysis of multiple analytes. Methods for makingsuch channels include nanoimprint lithography, photolithography,electron beam lithography, interference lithography, shadow masking,holographic lithography, ion beam lithography, and other methods knownto those of skill in the art.

Channels are suitably channels of square or rectangular cross-section(as shown in, e.g., FIG. 13 ), but may be of circular, ovoid, orirregular cross-section, as dictated by the needs of the user or byprocess constraints. The cross-section of a channel may vary along oneor more dimensions.

Nanoparticles, fluorophores, and the like may also be disposed withinthe channels. Moietites capable of interacting with a macromoleculedisposed within (or translocated through) a nanochannel may be disposedwithin the channels so as to give rise to devices capable of generatinga signal based on the interaction of a part of a macromolecule with anitem disposed within a channel.

Channels may also include one or more inlets or outlets. Such featuresmay allow for access to the channel from the side, from above, frombelow, or in essentially any orientation. Devices having channels andother fluidic elements disposed in two or three dimensions are withinthe scope of the claimed invention, and channels are suitably in fluidcommunication with one or more inlets, outlets, or both.

Base Substrate

The base substrate is composed of any substrate material that issemiconducting, insulating, or conducting, and is suitably capable ofbeing bonded to the transparent substrate through the bottom thin film,the top thin film, or both.

The base substrate need not be transparent to the electromagneticfrequencies of interest. While silicon is especially suitable, othermaterial choices include SiGe, Ge, strained silicon, GeSbTe, AlGaAs,AlGaInP, AlGaN, AlGaP, GaAsP, GaAs, GaN, GaP, InAlAs, InAlP, InSb,GaInAlAs, GainAlN, GaInAsN, GaInAsP, GalnAs, GaInN, GaInP, GaSb, InN,InP, CdSe, CdTe, zinc selenide (ZnSe), HgCdTe, ZnO, ZnTe, zinc sulfide(ZnS), aluminum, aluminum oxide, stainless steel, Kapton™, metal,ceramic, plastic, polymer, sapphire, silicon carbide, silicon oninsulator (SOI), astrosital, barium borate, barium fluoride, sillenitecrystals BGO/BSO/BTO, bismuth germanate, calcite, calcium fluoride,cesium iodide, FeUNbO₃, fused quartz, quartz, fused silica, glass, SiO₂,gallium, gadolinium garnet, potassium dihydrogen phosphate (KDP), KRS-5,potassium titanyl phosphate, lead molibdate, lithium fluoride, lithiumiodate, lithium niobate, lithium tantalate, magnesium fluoride,potassium bromide, titanium dioixde, sodium chloride, tellurium dioxide,zinc selenide, spin-on glass, UV curable materials, soda lime glass, anycompound above in an hydrogenated form, stoichiometric variations of theabove compounds, and the like, and any combinations thereof.

A substrate's thickness is suitably between about 0.01 mm to about 5 mm.The thickness can also be between about 0.1 mm and about 1 mm.

While a variety of labels may be used to analyze bodies of interest,light-emitting labels are well-known in the art and are consideredespecially suitable for use with the claimed invention Light emittinglabels used to analyze the bodies of interest are typically excited bymeans of fluorescence, luminescence, chemi-luminescence,phosphorescence, and the like; fluorescence is a commonly-used method.Suitable labels include organic fluorophores, quantum dots, metal dots,polymer beads, lanthanide chelates, nanoparticles, fluorescent beads,phosphorescent beads, semiconductor nanoparticles, dendrimers, molecularantennae, and the like, and any combination thereof. TOTO-3 is anexemplary fluorophore; other fluorophores may be used.

Targets for analysis suitably include molecules, macromolecules, singlestranded DNA, double stranded DNA, single stranded nucleic acidpolymers, double stranded nucleic acid polymers, RNA, polymers,monomers, enzymes, proteins, peptides, conjugate macromolecules,self-assembled macromolecules, pieces of cellular components,organelles, viruses, and the like and any combination thereof. Thepresent invention is considered especially suitable for use in DNAanalysis.

The present invention also provides methods of reducing the backgroundsignal of an analysis device, the methods including disposing a bottomthin film on a base substrate, transparent substrate, or both, the basesubstrate further defining at least one boundary of a channel; thebottom thin film being capable of reducing the signal of the channelemitted at a particular wavelength of electromagnetic radiation.

The wavelength of the excitation light is in the range of from about1000 nm to about 300 nm. Depending on the use of fluorescent labels, theexcitation wavelength may be chosen for optimal excitation of the label.For example, TOTO-3 labels are suitably excited by light in the red(e.g., 635 nm) range, and the signal that may be detected from suchexcited labels may be sent through a band-pass filter (665-705 nm) toremove reflected excitation light.

Bonding

The bonding process can be any suitable process that bonds thetransparent and the base substrates. In some embodiments, the bondingprocess is anodic bonding. Other bonding processes include, but are notlimited to: fusion bonding, thermal bonding, direct contact bonding,plasma-activated bonding, direct oxide bonding, polymer bonding,metal-metal bonding, thermo-compression bonding, eutectic bonding,chemically-activated bonding, ultrasonic bonding, dielectric polymerbonding, adhesive bonding, van der Waals bonding, and any combinationthereof.

EXAMPLES AND NON-LIMITING EMBODIMENTS Example 1

FIG. 18 shows a series of fluorescent images taken of the edge of theconfined channel, showing both the channel and the bonded regions. Theexcitation wavelength is red light (635 nm), and the detected signal ispassed through a band-pass filter (665-705 nm) to remove any reflectedexcitation light. As the silicon oxide thickness was increased, thebackground in the region where the transparent substrate and basesubstrate are bonded through the thin film produced an elevated amountof background in the wavelength region above 635 nm, whereas the channelregion maintains low background. It should be noted that the backgroundlevel measured with green light (532 nm) and blue light (473 nm) showedno variation with silicon oxide thickness. In this example, the siliconoxide was deposited using PECVD and the channel was filled with air.Images were taken with an EMCCD camera.

FIG. 18 thus illustrates the challenges posed by using a thin film layerthat produces a background signal when exposed to radiation that mayalso be used to elicit emission from a particular label. As shown inFIG. 18 , the device with SiO_(x) thin film produces a comparativelyhigh background level across a range of wavelengths, which poses tousers who might seek to analyze signals from labeled samples that emit(when exposed to excitation radiation) radiation in the same wavelengthas the background signal from the device. Put another way, the SiO_(x)device illustrated in this figure has a comparatively low signal/noiseratio, which would pose challenges for users seeking to pick out andanalyze labeled samples against the comparatively high background signalfrom the device.

The higher background level makes detection of weak signals from bodiesof interest close to the edge in the channel difficult or impossible.This is particularly problematic when the channel width is very narrow(approaching the wavelength of the excitation radiation or less, as isthe case when the channels are nanochannels), in which case the labeledbody of interest must have sufficient signal strength to overwhelm thebackground. However, as previously stated, removing the silicon oxidethin film to reduce the background will result in quenching of thelabeled bodies.

Example 2

FIG. 19 shows the same experiment as FIG. 18 , except that the siliconoxide thin film was replaced with a silicon nitride thin film. Siliconnitride was chosen as it is a dielectric material commonly used in thesemiconductor industry, and thus widely available in most semiconductorfoundries. In this example, there is no associated increase inbackground with nitride thickness.

FIG. 19 depicts a series of fluorescent images taken of the edge of theconfined channel, showing both the channel and the bonded regions. Theexcitation wavelength is red light (635 nm), and the detected signal ispassed through a band-pass filter (665-705 nm) to remove any reflectedexcitation light. As the silicon nitride thickness is increased, thebackground in the region where the transparent substrate and basesubstrate are bonded through the thin film shows no apparent increase ordecrease. The background level measured with green light (532 nm) andblue light (473 nm) showed no variation with silicon nitride thickness.In this example, the silicon nitride was deposited using PECVD and theconfined channel was filled with air. Images were taken with an EMCCDcamera.

Example 3

In this example, illustrated by FIG. 8 , double stranded human geneticDNA labeled with an intercalating dye (TOTO-3) was flowed in fluidthrough confined channels of various widths with a 58 nm SiO_(x) thinfilm. As the widths of the channels decreases, the DNA becomes lessvisible due to the high background levels from the regions where thebase substrate is bonded to the transparent substrate through theSiO_(x) thin film.

FIGS. 20A-B shows (a) a fluorescent image of DNA in confined channels ofvarious widths. The boundary between the channels and the bonded regionsare clearly visible due to the high background generated in the bondedregions, and (b) a fluorescent image of DNA in channels of width 100 nm.At this width, the DNA is barely visible due to the backgroundoriginating from the bonded regions (i.e., regions where one substrateis bonded to another). The background appears to be uniformly high dueto the very narrow widths of the nanochannels. FIG. 20C shows aschematic of the fluidic chip from which images (a) and (b) wereacquired. The SiO_(x) was deposited to a thickness of 58 nm over anetched silicon substrate using PECVD, and the transparent glasssubstrate composed of Schott Borofloat 33™ was anodically bonded to theSiO_(x) covered silicon. The TOTO-3 labeled DNA excited with red light(635 nm), and the detected signal was passed through a band-pass filter(665-705 nm) to remove any reflected excitation light.

As shown in the figure (e.g., FIG. 20B), the SiO_(x) thin film resultsin a device having comparatively high background signal (at the relevantwavelength) relative to the labeled sample. This relatively highbackground renders difficult the detection of weak signals from bodiesof interest (e.g., labeled DNA) close to the edge in the channel. Thisphenomenon is particularly acute when the channel width is very narrow,such as when the width approaches the wavelength of the excitationradiation or even less, as is the case when the channels are channels ofnanoscale width. In these instances, the labeled body of interest musthave sufficient signal strength to overwhelm the background, but theremay be limits on the number and brightness of the labels that can beplaced on the body of interest, as well as limits on the intensity ofthe radiation that can be used to excite the labeled body. Further, asexplained elsewhere herein, removing the silicon oxide thin film toreduce the background may then result in quenching of the labeledbodies, making analysis more difficult.

Example 4

In this example, shown in FIGS. 21A-C, DNA labeled with an intercalatingdye (TOTO-3) is flowed in fluid through confined channels of variouswidths with a 58 nm SiN_(x) thin film. As the widths of the channelsdecreases, the DNA remains visible, as the background levels do notincrease in the regions where the base substrate is bonded to thetransparent substrate through the SiN_(x) thin film, as compared withthe SiO_(x) thin film in FIGS. 20A-C.

FIG. 21A shows at section (a) a fluorescent image of DNA in confinedchannels of various widths. Unlike FIG. 20A, the channel boundaries arenot visible due to the low background. FIG. 21B illustrates afluorescent image of DNA in channels of width 100 nm. The SBR of thelabeled DNA is significantly higher than that shown in FIG. 20B. FIG.21C is a schematic of the enclosed channel chip from which chip images(a) and (b) were acquired.

In this non-limiting embodiment, the SiN_(x) was deposited to athickness of 58 nm over an etched silicon substrate using PECVD.Transparent glass substrate composed of Schott Borofloat 33™ wasanodically bonded to the SiO_(x) covered silicon substrate. TOTO-3labeled DNA was excited with red light (635 nm), and the detected signalwas passed through a bandpass filter (665-705 nm) to remove anyreflected excitation light.

Comparing the SiN_(x) thin film (FIGS. 21A-C) to the SiO_(x) thin film(FIGS. 20A-C) also serves to highlight another aspect of the claimedinvention. As shown in FIGS. 20A-C and FIGS. 21A-C, a SiN_(x) thin film(as compared to a SiO_(x) thin film) allows the fluorescently labeledmolecules under study to fluoresce when illuminated by excitationradiation, rather than the molecules being quenched and at leastpartially losing their ability to emit radiation of an emissionwavelength.

Thus, in some embodiments, one or more of the thin films is selected forits ability to reduce the background signal of the analysis device(compare FIG. 18 —illustrating the background signature for a sampledevice using SiO_(x) as a thin film—with FIG. 19 —showing the backgroundsignature for a sample device using SiN_(x) as a thin film). The thinfilm may further be chosen for its ability to allow a fluorescentlylabeled target to fluoresce when excited without quenching that label'sfluorescence (compare FIG. 20B—illustrating the quenching effect thatthe substrate may exert on a fluorescently labeled sample with FIG.21B—illustrating the lack of quenching present with a SiN_(x) thinfilm).

Without being bound to any particular theory, a particular thin filmmaterial may shield the fluorescent molecules from radiation that may bereflected from the substrate (or other source) during the fluorescentmolecules' exposure to excitation radiation. Also without being bound toany particular theory of explanation, the thin film material mayaccomplish its reduction of the background signal from the device byshielding or absorbing radiation of a particular wavelength that may bereflected from the substrate during the fluorescent molecules' exposureto excitation radiation.

While the disclosed, non-limiting embodiments highlight the advantagesof the claimed invention during analysis of TOTO-3—labeled DNA excitedwith red light (635 nm) disposed within a device having a SiN_(x) thinfilm and Si and Borofloat 33™ substrates, the invention is not limitedto this sample embodiment. As described elsewhere herein, the substratesand thin films of the claimed invention may include many differentmaterials, and the optimal combination of thin film, label/fluorescenceand substrate for a particular method of analysis will be easily foundby the user of ordinary skill. In some embodiments, the invention allowsfor a user—by selection of an appropriate thin film—to reduce thebackground signal of a device, to reduce the quenching that a device mayeffect on fluorophores disposed within the device.

As explained elsewhere herein, quenching or otherwise limiting theability of a fluorophore or other label to reflect or emit radiation maybe undesirable because such quenching limits the ability of the user toresolve the target against the background. By avoiding (or at leastreducing) such quenching, the present invention enhances the ability ofthe user to resolve the presence or position of such labels against thebackground. SiN_(x) is one material that does not quench fluorophores'ability to fluoresce (while also reducing the background of the analysisdevice, as shown in FIG. 7 and FIGS. 21A-C). Other materials that reducethe background while also minimizing quenching will be easily identifiedby the user of ordinary skill in the art.

In some embodiments, the device includes a channel or chamber disposedin a chamber material (e.g., SiN_(x)) that is itself a comparativelylow-background material that minimizes the quenching of fluorophoresthat are exposed to excitation radiation while disposed within thechamber. Such chambers may be formed in the material by, for example,disposing a sacrificial material within the chamber material andselectively removing the sacrificial material so as to leave behind achannel that substantially conforms to the removed sacrificial material.

Exemplary Embodiments

FIG. 1 depicts a schematic view of a device according to the claimedinvention. The device in that figure includes two substrates, A and B,bonded to one another. Substrate A has a thickness of DA, and substrateB (the upper of the two substrates) has a thickness of DB.

As shown in the figure, a port (which may be an inlet or outlet) extendsthrough substrate A or B so as to place the nanoscale structures on thedevice in fluid communication with the environment exterior to thedevice. In some embodiments, the port extends through the entirety ofthe device, and in some embodiments allows introduction (or removal) offluid from

Interconnects—which may be microscale channels or conduits—place theport in fluid communication with the front-end (FE) structures locatedon the device. A port may extend through the full thickness of asubstrate or partially through the substrate's thickness.

The FE structures may act to partially extend or elongate amacromolecule (such as DNA) that may be analyzed in the device.Macromolecular elongation is further explained in U.S. application Ser.No. 10/484,293, the entirety of which is incorporated herein byreference. Suitable FE structures are described elsewhere herein, andcan include crow-form channels, eagle-form channels, pillars, posts, andother structures that may act to elongate a tangled or folded body thatis flowed against or through the structures. Such structures aresuitably patterned on one or both of the substrates.

Also shown in FIG. 1 is a nanochannel array device, which device may befabricated on substrate A, substrate B, or some combination thereof(e.g., some parts of the array are fabricated on substrate A, and otherparts being fabricated on substrate B). Suitable nanochannels andmethods for analyzing macromolecules disposed in nanochannels are alldescribed in U.S. application Ser. No. 10/484,293, the entirety of whichis incorporated herein by reference.

In some embodiments, the analysis methods include exposing a DNA targetto one or more labels, translocating the DNA target through a deviceaccording to the present application, and interrogating (e.g.,optically) the DNA target for the presence (or absence) of the label.Fluorescent dyes and related instruments are considered suitable forsuch an analysis.

The nanochannel array may include one or more nanochannels, which may bearranged in parallel, serpentine, converging, diverging, zig-zag,curved, or other such patterns, as shown in the attached figures.

In one non-limiting embodiment, the nanochannel array includes a singlenanochannel that doubles back on itself, as shown in FIGS. 10A-C. Ananochannel may be of constant or of varying cross-section, and multiplenanochannels present on the same device may be of different sizes.

The devices shown in FIG. 1 also, in some embodiments, include aback-end (BE) structure that may be disposed between the nanochannelarray and a port, outlet, or other conduit. The BE structure is suitablyof a configuration suitable for a FE structure (described elsewhereherein), and may include one or more channels, pillars, obstacles, andthe like. Such BE structures suitably assist in transporting a target(e.g., a macromolecule) from a nanochannel analysis region to aninterconnect or other conduit. The BE may assist in transporting atarget from a nanoscale (e.g., nanochannel) environment to anenvironment that contains larger (micron-sized, or larger) structures.

Devices according to FIG. 1 may be of varying dimensions. The devicessuitably have a length (L) of from about 0.1 mm to about 100 mm, a width(W) of from about 0.1 mm to about 100 mm, and the substrates (shown as Aand B) suitably have a thickness in the range of from about 10 nm toabout 10 mm. A given device may have from 1 to about 1000 independentnanochannel array devices, and a device may even have from about 2 to500 individual ports. The optimal number of arrays and ports will dependon the needs of the user.

FIG. 2A depicts an exemplary nanodevice chip, with red arrows depictingthe direction of the views of cross sections of the device illustratedin FIG. 2A through 5B. FIG. 2B depicts an exemplary, non-limitingfabrication scheme according to the claimed invention. In thisembodiment, fluidic elements are formed on the lowermost substrate andthe lowermost substrate is then bonded (e.g., anodically bonded) to theupper substrate, which upper substrate can be glass or a suitabletransparent material.

FIG. 3A shows an exemplary fabrication scheme, wherein one substrate(either substrate A or B) having channel elements etched thereon iscoated either by growth of thermal oxide or conformal deposition methodssuch as atomic layer deposition (ALD) on the surface of the substrate,which is then bonded to a second substrate through fusion or anodicbonding. FIG. 3B shows a non-limiting fabrication scheme for a substratewith etched channel elements in the upper substrate, which uppersubstrate is suitably transparent glass and can be anodically bonded toa lower (e.g. silicon) substrate that has a film (e.g., silicon dioxide)thermally grown or otherwise deposited throughout the entire surface oronly the bonded surface. Channels can be etched onto both substrates;when the substrates are bonded to one another, multiple channels result,or—if the channels on the substrates are in registration with oneanother—a single channel may be formed (FIG. 13 ).

FIG. 4A depicts an example fabrication scheme for two substrates(substrate A or B), with channel elements etched into both substrates,and then a subsequent step of coating the bottom substrate either bythermal oxide growth or conformal deposition methods such as ALD. Thesubstrates are then bonded together through fusion or anodic bonding,with at least portion of the channel on opposing substrate surfacesoverlapping. FIG. 4B depicts another non-limiting fabrication scheme, inwhich a layers of coating are deposited on both substrates, with channelelements then being etched into the coating layer and into the lowersubstrate, the substrates then being bonded together through fusion oranodic bonding, with at least portion of the channels on opposing bondedsurfaces overlapping.

FIG. 5A depicts an exemplary nanodevice chip with arrows depicting thedirection of the aerial view of the channel patterns on the device,illustrated at FIG. 5A to FIG. 11C.

FIG. 5B depicts non-limiting layouts for a 4 port example embodiment anda 2 Port example embodiment configuration. The arrows indicate thedirection of the sample (e.g., DNA) flow. The sample need not flow inthe direction shown, and the flow direction may be stopped or evenreversed as desired.

This embodiment depicts one suitable relationship between the ports, theinterconnect regions, FE and BE regions, and a nanochannel array. Byarranging these components in such a manner, the device enablesmanipulation of a target (e.g., DNA or another macromolecule) across awide range of length scales, from the centimeter scale (10″ m) of theinlet port to the millimeter (10″ m) scale of the interconnects andFE/BE regions, on down to the nanometer (10⁹ m) range of thenanochannels in the nanochannel analysis region. While the analysisregion is labeled “nanochannel array region” in FIG. 5B, the analysisregion may include a single nanochannel, or nanochannels that are notarranged in an array-like formation.

FIG. 6A depicts an example embodiment of a multi-port device design. Thedesign in FIG. 6A has 16 ports, including 8 independent 2 port devices.FIG. 6B depicts a design having 16 ports, including 4 independent 4 portdevices. These embodiments allow the user to simultaneously analyzemultiple, different targets.

FIG. 7A depicts a multi-stage branched channel array. In this example,there are 5 stacked arrays of channels, the channels havingprogressively smaller cross sectional dimensions, and the channels beingconnected by 5 levels of forks bridging the microfluidic inlet channelsand the nanochannel analysis region, located at the bottom of FIG. 7A.The distance between the forks is suitably about 50 microns, and the twosmaller channels suitably half the cross-sectional area of the originalchannel at each branch.

As shown, at each fork, the channel is divided into two smallerchannels. The branch angle is suitably between about 30 and about 60degrees, although it can range from about 0 to about 90 degrees, and Mis suitably from about 0.4 to about 0.6 W. As a matter of nomenclature,embodiments—such as the device shown in FIGS. 7A-E—that have a channelsplit by a pointed or triangular fork structure are known as “crow”devices or “crow” channels, which are described in more detail elsewhereherein.

A target (e.g., a fluid-borne macromolecule) may pass through from 1 to15 or more divided channels during analysis, and the length (L) of eachbranch channel can vary from about 5 to about 80 micrometers. The usermay alter the number of forks and the relative size of a secondarychannel to a primary channel so as to enable controllable movement for atarget moving from the comparatively large inlet port on to thenanoscale nanochannel analysis regions of the claimed devices.Multi-stage divided-channel structures (FIGS. 7A-E) may be used.

FIG. 7B illustrates a Scanning Electronic Microscopy (SEM) image of abranched forks interconnecting two arrays of channels of differentsizes. FIG. 7C shows a cartoon view of a branching fork design having acomparatively sharp split at the fork, although the angle at the forkcan be from about 0 to about 90 degrees.

FIG. 7D is an image taken from a video of fluorescently labeledmolecules moving inside the channels, highlighting the channels andinterconnecting forks. FIG. 7E is a fluorescent image of singular,comparatively long genomic DNA molecules moving from large channels intobranched narrower channels, where the molecules are elongated. The sharpsplit at the fork is seen, outlined by singular DNA molecules.

In FIGS. 7A-E, multiple “crow” structures are used, such that amacromolecule or other target that enters the interconnect region shownat the top of the figure will pass through 5 (or more) forks/splitsbefore the target enters the nanochannel array region shown at thebottom of the figure.

As discussed, the distance between the forks can be about 50 microns(though the separation distance can be greater or smaller than 50microns), and the smaller channels that emerge from each fork are eachabout half the size of the original channel at each branch. Thus, thetotal cross-sectional area available to a fluid contained within thesecondary (or “branch”) channels is approximately equal to thecross-sectional area of the primary (or “trunk”) channel. By maintainingalong the length of a branched channel device an essentially constantcross-sectional area available for fluid flow, the disclosed devicesminimize the changes and disruptions in flow fields that can result fromchannels of narrowing or broadening cross-sectional area.

FIG. 8A depicts a schematic of a second design for an alternative,multi-level branched, interconnected channel array. FIG. 8B shows aScanning Electronic Microscopy (SEM) image of one of the branched forksinterconnecting two arrays of channels of different sizes.

FIG. 8C shows the branching fork design having a more rounded orcontoured bend around the fork. FIG. 8D shows an image taken from videoof fluorescently labeled molecules moving inside the channels,highlighting the channels and interconnecting forks, and FIG. 8E shows afluorescent image of singular long genomic DNA molecules moving fromlarge channels and being elongated into branched narrower channels. Twodifferent levels of contoured bends at the forks can be seen outlined bysingular DNA molecules.

For the fluorescent images were, the DNA sample consisted of male humangenomic DNA stained with an intercalating dye (YOYO-I) at a ratio of 5base pairs per dye molecule. The DNA was suspended in 0.5×TBE buffer ata concentration of 5 ng/uL. DNA was flowed into the nanochannels usingeither capillary flow or via electric field with an applied voltage inthe range of 0-50V. Excitation of the sample was performed using a lightemitting diode and the fluorescence emission was collected through a 60×objective and detected using an electron multiplying CCD camera.

FIGS. 8A-E thus depict channels according to the “eagle” configuration.As shown, the fork that splits the primary channel into branch channelsis suitably a rounded structure, such as rounded pillar. The diameter oreffective cross-section of the fork is suitably such that the edge ofthe fork extends into the channel that precedes the fork.

Without being bound to any particular theory, in this configuration amacromolecule (or other target) that, in a channel, follows an electricfield's path (e.g., from an applied gradient) will be more likely toenter the center of a following channel rather than the edge, as shownin the figure. Thus, targets will be less likely to enter certainchannels over others in the branched network, and the result is a moreuniform loading of the nanochannels in the nanochannel array.

In one example embodiment, M is 0.3 to 0.7 times W, and X is 0.2 to 0.5times W. The number of forks the a target may traverse before reachingthe nanochannel array can be from 2 to 15, and the length of each branchchannel (L) can vary from 5 to 80 microns.

In some embodiments, multiple “eagle” structures are used, and thenumber of forks in each eagle structure is 5 before the target willenter the nanochannel array region. The distance between the forks inthis non-limiting embodiment is 50 microns (though this distance can begreater or smaller than 50 microns), and the two smaller (branch)channels are half the original channel, such that the totalcross-sectional area available for fluid flow is the same at any planealong the length of the device.

FIG. 9A shows a schematic of another design, showing a combination ofbranched channels and post arrays. In one embodiment, a branched channelarrays interconnect with one another, and within the channels are arraysof posts. FIG. 9B shows a Scanning Electronic Microscopy (SEM) imageshowing dense round shaped post arrays embedded within channels.

FIG. 9C shows a schematic of a design having branched channels and postarrays. In one embodiment, with multi-level branched channels connectingto one another, there are arrays of diamond shapes posts of graduallyreduced sizes and increased density. FIG. 9D shows a Scanning ElectronicMicroscopy (SEM) image showing dense post arrays embedded withinchannels interconnecting with downstream channels of smaller sizes. FIG.9E shows a fluorescence image of comparatively long genomic DNAmolecules moving within a post array with channels.

FIG. 10A depicts a design having a single long nanochannel arranged in acontinuously connected, serial set of parallel nanochannels in aserpentine configuration; only a single set from an array of thisconfiguration is shown here. FIG. 10B shows a Scanning ElectronicMicroscopy (SEM) image showing a boxed area of such a serpentineconfigured nanochannel etched into a silicon substrate, showing theturns of the channel. FIG. 10C shows a fluorescence image of a genomicDNA molecules moving within the nanochannel and making a 180 degreeturn.

This configuration addresses, inter alia, the challenge of visualizingan elongated or elongating macromolecule in a single field of view.Because macromolecules may be very long, a channel of sufficient lengthto elongate a macromolecule may be longer than the width of ahigh-magnification microscope's field of view. This in turn prevents theuser from visualizing the entire macromolecule in a single field ofview.

A device that has a nanochannel in a serpentine or switch-back patternas shown in FIGS. 10A-C, however, increase the length of channel thatfits within a single field of view and thus enables the user to view anelongated macromolecule in a single field of view. Alternatively, such adevice enables a single field of view to cover a substantial portion ofthe elongated macromolecule. Serpentine, switch-back channels alsoincrease the residence time of a translocating macromolecule within asingle field of view.

FIG. 11A depicts multiple, long nanochannels arranged in a continuouslyconnected serial set of parallel nanochannels, the difference from theprevious figure being that each channel stage has a progressivereduction in its channel width, from 1000 nm down to 100 nm. FIG. 11Bshows a Scanning Electronic Microscopy (SEM) image, with a boxed area ofone set of such serpentine configured nanochannel etched into a siliconsubstrate, showing the gradually reduced width of the channels frombottom to top and then the comparatively wide channel outlet.

FIG. 11C shows a set of time-lapse video frames (each panel represents adifferent point in time) that track the fluorescent image of a singlegenomic DNA molecule moving within channels described in FIGS. 11A-C,the molecule having a progressively stretched length as it entersnanochannel regions of smaller and smaller sizes. To act as a control orreference standard, an image of a stationary molecule is shown, and thelength of the stationary molecule is shown as outlined between the twodashed lines drawn across all of the panels of assembled frames. Turningto the various image panels, the uppermost panel shows a bright fieldoptic image of the actual chip pattern, and the fifth panel shows afluorescent image of the DNA molecule turning a corner.

FIG. 12(a) shows a Scanning Electronic Microscopy (SEM) image showinganother non-limiting design, this design including an array of parallel,non-straight nanochannels arranged in a zig-zag pattern. FIG. 12(b)shows an image of fluorescently labeled DNA molecule stretched insidethe zig-zag shaped channel. FIG. 12(c) shows a Scanning ElectronicMicroscopy (SEM) image of an arbitrary nanochannel pattern (the letters“BNM”), with the channels in the pattern all having an essentially equalchannel width. FIG. 12(d) shows a Scanning Electronic Microscopy (SEM)image showing two sets of perpendicular nanochannels intersecting witheach other, the overlapping region appearing as a dense, rounded postarray.

Fabrication

The fabrication process may include fabricating fluidic features on asubstrate surface, and then bonding the substrate surface to a secondarysubstrate to form an enclosed fluidic device accessible by the ports.Alternatively, the fabrication may include fabricating fluidic featureson a substrate surface, and fabricating fluidic features on a secondarysubstrate surface, and then bonding the two substrate surfaces togetherto form an enclosed fluidic device accessible by the ports.

Substrate material can include, but is not limited to: silicon, silicondioxide, silicon nitride, hafnium oxide, quartz, glass, fused silica,metal, aluminum oxide, metal, ceramic, polymer, plastic, dielectrics,SiGe, GaAs, GaAlAs, ITO, and the like. In one example embodiment, atleast one of the substrates must be transparent to UV, visible, andinfrared electromagnetic radiation.

In one example embodiment, the substrates are wafers of glass, silicon,and or quartz, and after bonding, the chips are obtained by dicing thebonded wafers. In one example embodiment, the fluidic elements arefabricated using methods known to the semiconductor, MEMS andmicrofluidic industry, including, but not limited to: photolithography,plasma etching, material deposition, wet etching, bonding, and anycombination thereof.

In one example embodiment, the nanochannel array, front-end/back-end,and interconnects are patterned (e.g., via photolithography) onto asubstrate (such as silicon), after which the patterns are transferredinto the silicon by etching. A variety of patterning and etching optionsare possible:

Patterning may be accomplished by, e.g., photolithography, nanoimprintlithography, embossing, interference lithography, near field holography,contact printing, extreme UV lithography, electron beam lithography orany combination thereof.

For these patterning options, the use of a hard or soft mask can aid inthe pattern transfer to the substrate. These masks include, but are notlimited to: anti-reflection coatings, silicon oxide, silicon nitride,dielectrics, metals, organic films, combinations thereof, and the like.For all of these patterning options, various intermediate patterntransfer methods could be used, including, but not limited to: lift-offprocesses, shadow evaporation, growth, deposition, combinations thereof,and the like.

Etching options include—but are not limited to—chemical etching, wetetching, etching with KOH, etching with TMAH, etching with HF, etchingwith BOE, ion etching, reactive ion etching (RIE), plasma etching,plasma assisted etching, inductively coupled plasma (ICP) etching, boschetching, patterned oxide growth in silicon (such as LOCOS) and removalwith a wet etch, combinations thereof, and the like.

Patterning Order

In one example embodiment, the nanochannel array and Front-end/Back-end(FE/BE) are patterned and etched simultaneously, the interconnects beingpatterned later. However, this need not be the case, and the order ofpatterning these fluidic elements can vary.

The nanochannel array can be patterned by interference lithography, andthe front-end/back-end patterned by photolithography in a separate step.In another embodiment, the nanochannel array, front-end/back-end, andinterconnects is suitably patterned in a single step usingphotolithography or nanoimprint lithography. In another embodiment,patterning technologies capable of transferring variable-depth featuresinto the substrate such as nanoimprint or embossing are used to allowthe interconnect, front-end/back-end, and nanochannel array to havedifferent depths, all with a single patterning step.

Ports

Ports are suitably patterned by photolithography, and then etched withan etch process such as a deep silicon etch (“Bosch Etch”). However, avariety of fabrication options are available for fabricating ports. Anon-limiting list of such options includes RIE, ICP etching, plasmaetching, laser drilling, laser ablation, sand blasting, drilling, wetetching, chemical etching, water drilling, ultrasonic drilling, and anycombination thereof.

The port suitably has a width (diameter) of 5 to 5000 microns, and thedepth is the thickness of the substrate that it goes through. In oneexample embodiment, the port has a width (diameter) that ranges from 50to 2000 microns.

Bonding

In one example embodiment, the fluidic elements of the device arecompleted by anodically bonding a patterned silicon substrate with anun-patterned glass wafer.

In one example embodiment, the glass wafer to be anodically bonded canbe Pyrex 7740, Schott Borofloat 33™, Hoya SD2™, or any glass withsimilar thermal expansion characteristics. Other options are suitable,including (but not limited to) fusion bonding, thermal bonding, chemicalbonding, quartz-quartz bonding, glass-glass bonding, polymer bonding,solvent bonding, adhesive bonding, combinations thereof, and the like.

Bonding conditions—anodic and otherwise—will be easily optimized by theuser of ordinary skill in the art. As one non-limiting example, siliconand Borofloat™ glass may be anodically bonded together using a voltageof 400V, a temperature of about 350° C., applied for 5 min. Anodicbonding voltages may range, for example from about 200 V to about 800 V,temperatures suitably range from about 200° C. to about 400° C., andapplication time from about 1 to about 100 min.

Fluidic Element Surfaces

A variety of materials can compose the surface of the fluidic elements,including, but not limited to: silicon, silicon dioxide, siliconnitride, hafnium oxide, quartz, glass, fused silica, metal, aluminumoxide, metal, ceramic, polymer, plastic, dielectrics, SiGe, GaAs,GaAlAs, ITO, organic molecules, self-assembled monolayers,self-assembled multi-layers, combinations thereof, and the like. In oneexample embodiment, the fluidic elements will have a dielectric surface;in some embodiments, fluidic elements will have a silicon dioxide and/orglass surface.

Fabrication Example

In one non-limiting embodiment, fluidic elements (nanochannel array,front-end/back-end, interconnects, and ports) after bonding have asilicon dioxide or/and glass surface, such that fluid disposed withinthe resultant device contacts only silicon dioxide or/and glass. Thissurface is formed by depositing a film of silicon dioxide over theetched silicon surface after the patterning and etching of thenanochannels, front end/back-end, interconnects, and ports.

An oxide is deposited by atomic layer deposition (ALD) on the patternedand etched silicon substrate, and has a thickness from about 1 nm toabout 5000 nm. This silicon wafer is then anodically bonded to a glasssubstrate.

The silicon dioxide surface serves several useful purposes. First, thesilicon dioxide provides an insulated film which is useful when anelectric field is used to drive the movement of DNA in the fluidics, andone of the substrates is silicon.

The silicon dioxide also provides a surface that can be functionalizedand or passivated as required by the application. The layer furtherallows the nanochannel cross-section to be modified (tailored) to thedesired size when the oxide is grown or deposited on the preexistingetched nanochannel.

In one example, a 200 nm wide and 150 nm deep nanochannel is reduced to100 nm wide, and 100 nm deep when 50 nm of conformal oxide is depositedover the nanochannels. In this way, application of a coating to analready-formed fluidic element (e.g., a groove or trench) allows theuser to controllably build up the boundaries of that element so as toreduce the cross-section of that element that is available to fluidflowing therein.

Silicon dioxide is also transparent to a wide spectrum ofelectromagnetic radiation, including UV, visible and infrared light.

There is a wide variety of fabrication options for forming fluidicchannels with a silicon dioxide and/or glass surface. These include (butare not limited to):

Thermal Oxide Growth on Silicon

If one of the substrates to be used is silicon, the silicon dioxidesurface can be achieved by growing the oxide using the silicon surfaceas a source of silicon. Examples include, but are not limited to: drythermal oxide growth, wet thermal oxide growth. This appliesirrespective if all, some, or none of the fluidic elements are to bepatterned and etched in the silicon. Non-limiting, silicon-basedembodiments are set forth in the attached figures.

Deposited Oxide on Silicon, Glass, or Quartz

The oxide can be deposited on one or both of the substrates. Examplesinclude, but are not limited to: PECVD, CVD, LPCVD, thermal evaporation,spin-on glass, e-beam evaporation, sputtering, ALD and any combinationthereof. Representative examples are shown in, e.g., FIGS. 2-5 .

Etching Directly into Silicon Dioxide, Quartz, or Glass

Furthermore, a silicon dioxide or glass surface can be achieved byetching the fluidic elements directly into silicon dioxide or glass.This can be done by etching directly into the silicondioxide/quartz/glass substrate, or etching into a film of silicondioxide on a silicon substrate. See, e.g., FIGS. 2-5 .

Device Configuration

In FIG. 5 , the primary input and output ports opposite one another suchthat if an electric field were to be applied, the field strength wouldbe approximately equal in all of the nanochannels of the nanochannelarray. In one example embodiment, all three fluidic elements of thedevice: nanochannel array, frontend/back-end, and interconnects areincluded.

In another example embodiment, the front-end and/or back-end could beomitted, with the interconnects connected directly to the nanochannelarray. In another example embodiment, the interconnects could beomitted, with the front-end and/or back-end connecting directly to theports.

In another example embodiment, both the front-end and back-end and theinterconnects could be omitted, thus having the nanochannel arrayconnecting directly to the ports. In an example embodiment, the deviceis symmetrical so as to maximize the uniformity of the electric fieldstrength though the nanochannels in the nanochannel array when anelectric field is applied between the inlet and outlet ports.

In another example embodiment, the outlet of the nanochannel array leadsto an inverted frontend structure (called the back-end or BE), and theninto the interconnect channels as in FIG. 5(b). In another exampleembodiment, the outlet of the nanochannel array leads directly to theoutlet port (omitting the back-end and interconnects).

In another example embodiment, the outlet of the nanochannel array couldlead directly to an interconnect that leads to the outlet port (omittingthe back-end). In another example embodiment, the back-end could leaddirectly to the outlet port (omitting the interconnects).

2-Port Device

The 2 Port chip has one input in which the sample is loaded, and oneoutput in which it is subsequently removed. Sample movement is directlycontrolled via these two ports using forces such as electroosmotic,electrokinetic, electrophoretic, pressure, capillary, or any combinationthereof. This design has significant advantages including simple ease ofhandling for direct capillary sample loading. The design also minimizesthe number of ports, and thus maximizes the number of independentdevices allowable per chip.

4-Port Device

The 4-port device has two input (primary/secondary) ports, and twooutput (primary/secondary) ports. The principal advantage of this designover the 2-port chip is to provide the chip operator more degrees offreedom in controlling the sample movement through the nanochannelarray. Sample movement can be directly controlled via these four portsusing forces such as electroosmotic, electrokinetic, electrophoretic,pressure, capillary, combinations thereof, and the like. In thisapplication, the sample is flowed from the primary to the secondaryinlet port in a controlled manner, and once an item of interest isidentified, it can be translocated into the nanofluidic FE region bymodulating sample flow.

Gradient Front End and Back End

The Front-End and Back-End are characterized as the interfaces betweenthe microfluidic and the nanofluidic regions. The front end (FE)suitably facilitates the unraveling, elongation, and transition of theDNA from the microfluidic-scale interconnect region into thesmaller-scale nanochannel array. This is suitably accomplished byflowing the DNA through a network/array of densely patterned,progressively smaller (and more closely spaced) structures, whicheffects DNA elongation as the DNA approaches and then enters ananochannel or nanochannel. FE designs are suitably a variant of the“branched channel network” structure, which structure has severalattributes.

First, with each branch, the channel is split into two or more channels.In one embodiment, the total widths of the branching channels areapproximately equal to the original, such that the total cross sectionalarea remains approximately the same. In this manner, flow ratethroughout the branched channel network should remain approximatelyconstant.

Second, by progressively splitting, the branched network promotesuniform loading of DNA into the nanochannel array, i.e., there is nobiasing of a particular nanochannel, or set of nanochannels within thenanochannel array.

Further, the branched channel network presents progressively smallerfluidic channels that efficiently untangle and elongate very longsegments of DNA.

At a given branch point, the branching channels need not be the samewidth, length, or depth. Nor do they have to be parallel to each other,or uniformly distributed. Nor do the branched channels have to bestraight or linear in configuration. In some embodiments (FIG. 9 , forexample), to further enhance their ability to untangle the DNA, thebranch channels may contain pillar structures.

The FE fluidic structures are approximately 10-1000 nm deep, and up to10,000 nm wide. A channel (or a pillar or other obstacle) in a FEstructure can also have a depth of from about 100 to about 500 nm, oreven about 200 nm to about 300 nm. Structures (e.g., channels, pillars,and the like) can also have a width in the range of from about 1 toabout 10,000 nm, or from about 20 nm to about 5000 nm, or from about 50nm to about 1000 nm, or even from about 100 nm to about 500 nm.

Because the purpose of these structures is to gradually confine the DNAsample from the microfluidic environment to the nanofluidic environment,in one example embodiment these fluidic structures have a depth thatspans from 1000 nm to the depth of the final nanochannel, and a widththat spans from 10,000 nm to the width of the final nanochannel.However, this reduction in feature size in the FE structure need not bemonotomically decreasing, nor may it require a continuous variation infeature sizes. For example, a change in the feature sizes (depth andwidth) of the FE can be done in steps.

“Crow” Configuration

In the “crow” embodiments shown in FIG. 7 , the branched channel FEdesign includes a comparatively sharp fork (splitter) that splits thechannel into two new channels. The new channels can be the same size, orsmaller than the original channel. The branching angle can vary from 0to 90 degrees. The length of the branched channels can vary from 5 to500 microns. Each branching stage need not be the same length.

“Eagle” Configuration

The “eagle” design differs from the “crow” design. First, the fork isshaped as a rounded pillar. Second, the diameter of the pillar fork issuch that edge of the pillar protrudes into the channel that precedesit. The purpose behind this design is that a macromolecule (or othertarget) following an electric field path (or other gradient) will bemore likely to enter the center of the succeeding channel (rather thanalong the edge). In this way, targets are less likely to bias certainchannels over others in the branched network, and will instead result ina more uniform loading of the nanochannels in the nanochannel array. The“eagle” configuration (like the “crow” configuration) may suitablyinclude pillars disposed upstream, within, or downstream from thechannels.

Additional Embodiments

The nanochannel array forms the active region of the device. Here theDNA is analyzed. The patterning, width, depth, pitch, density, length,and area of the array can vary greatly. The nanochannels can be fromabout 10 nm to about 500 nm deep, with a width of about 10 to about 1000nm. The nanochannel widths and depths can remain constant through-outthe device, or vary, either along the channels, among the channels, orboth. The nanochannels can be separated by a distance anywhere from 10nm to 10 cm, can be anywhere from 0.1 micron to 50 cm in length, and thearray can span anywhere from 0.1 micron to 50 cm across. The channelscan be parallel, or non-parallel. They do not have to uniformlydistributed. They can be of identical length, or of differing lengths.They can be straight, or have turns and curves. They can be isolatedfrom one-another, or intersect.

Primary channels of the branched structures may be separated bydistances in the range of from about 1 micron to 50 microns, 100microns, 1000 microns, or 10 cm. The optimal pitch (spacing) betweenchannels will depend on the needs of the user, and can be identifiedwithout difficulty by those of skill in the art.

In one example embodiment the nanochannels are patterned in a parallelarray, with a depth of 20-500 nm, and a width of 20-800 nm. For aparticular device, the nanochannel's width and depth are constant. Thenanochannels are spaced 100 to 2000 nm apart, and are straight. Thelength of the nanochannels vary from 50 microns to 5000 microns.However, a variety of different nanochannel array embodiments can berealized, including embodiments where a nanochannel's width, depth, orboth may vary along the length of the nanochannel.

Interconnects

The interconnect fluidics can have a depth of 100 nm to 100 micron, anda width of 0.5 micron to 1000 microns. In one example embodiment, thedepth ranges from 200 nm to 20 micron, and the width ranges from 1micron to 50 microns.

Additional Description

In some embodiments, the present invention describes a fluidic deviceincluding a substrate (A) bonded to a secondary substrate (B), either orboth of which may be patterned. The fabrication process describes themicro- and nanofluidic elements that are confined by a bonding process,such as anodic bonding, between the silicon substrate to a glasssubstrate.

The active region of the chip is suitably located at the interface ofthe two substrates, where a single or multiple independent nanochannelarray devices are fabricated on one or both of the substrate surfaces.These devices are suitably in fluid communication with the environmentexterior to the chip via conduit ports extending through one or bothsubstrates.

The disclosed devices suitably include:

-   -   Nanochannel Region—The core device region: Here the        macromolecule (e.g., DNA) of interest is elongated, linearized,        imaged and analyzed.    -   Gradient Front-End (FE) and Back-end (BE)—An array of        interconnected branched channels with cross sectional dimensions        ranging from micron, submicron, or in the nanometers range. The        FE or BE can also include repeating micro- to nanoscale sized        structures such as posts, pillars, wells, grooves, and the        combination of the above, which structures interfaces        microfluidic and nanofluidic regions of the devices.    -   Interconnect—The microfluidic region: A network of microfluidic        channels that bring the sample of interest from the input ports        to the FE region, and provide a conduit for the sample to move        from the BE region to the output ports.    -   Ports: Holes suitably etched through the substrate(s), allowing        fluidic communication from the environment exterior to the        device to nanofluidic devices (suitably disposed between the        substrate A and B) inside the chip through a three dimensional        fluidic connection.

A variety of materials can compose the surface of the fluidic elements,including, but not limited to: Silicon, Silicon Dioxide, siliconnitride, hafnium oxide, quartz, glass, fused silica, metal, aluminumoxide, metal, ceramic, polymer, plastic, dielectrics, SiGe, GaAs,GaAlAs, ITO, organic molecules, self-assembled monolayers,self-assembled multilayers, or any combination thereof.

The present invention discloses devices with all fluidic elements have adielectric surface by atomic layer deposition (ALD), Pressure EnhancedChemical Vapor Deposition (PECVD), sputtering, thermo growth, or otherentropic or anisotropic material deposition methods. This step providesinsulation for electric field manipulation of biological molecules inthe fluidic elements as well as further reduction of the fluidic channelmanufactured by conventional fabrication methods.

The present invention also discloses nanofluidic element surfaces thatcan be functionalized and or passivated as required by the application,which surfaces can be transparent to a wide spectrum of electromagneticradiation, including UV, visible and infrared light.

The nanofluidic devices may also have multiple ports, and can includeinterfacing, progressively branched channel pattern design havingvarious specifications and angles, as shown in the included figures.

Devices with interfacing progressively branched channel patterns withvarious branching fork specification and angles. Various combinations ofbranching channels and post or pillar arrays may also be used tointerface between different regions of the disclosed devices, and mayalso be used as interfaces between channels having different widths.

1.-100. (canceled)
 101. A method of fabricating an analysis device,comprising: bonding a first substrate and a second substrate, at leastone of the substrates comprising at least one channel having a width inthe range of from about 10 nm to about 10,000 nm, the bonding givingrise to an enclosed conduit disposed between the substrates, theenclosed conduit being capable of transporting a fluid therethrough,wherein the enclosed conduit comprises a first front-end branchedchannel region and a nanochannel analysis region, wherein the firstfront-end branched channel region comprises at least a primary channelcharacterized as having a cross-sectional dimension of less than 10,000nm and at least two secondary channels, wherein the totalcross-sectional area of the at least two secondary channels isapproximately equal to the cross-sectional area of the primary channel,and wherein the nanochannel analysis region comprises at least onenanochannel characterized as having a cross-sectional dimension lessthan that of the primary channel, wherein the length of the at least onenanochannel is from 0.1 micron to 50 cm, and wherein the ratio of thecross-sectional dimensions of the primary channel to the at least onenanochannel is in the range of 100 to 10,000.
 102. The method of claim101, wherein the bonding comprises anodic bonding, thermal bonding, orany combination thereof.
 103. The method of claim 101, wherein the firstsubstrate, the second substrate, or both, comprises a dielectric and/ora semiconductor.
 104. The method of claim 101, wherein the firstsubstrate, the second substrate, or both, comprises silicon, SiGe, Ge,strained silicon, GeSbTe, AlGaAs, AlGaInP, AlGaN, AlGaP, GaAsP, GaAs,GaN, GaP, InAlAs, InAlP, InSb, GaInAlAs, GaInAlN, GaInAsN, GaInAsP,GalnAs, GaInN, GaInP, GaSb, InN, InP, CdSe, CdTe, zinc selenide (ZnSe),HgCdTe, ZnO, ZnTe, zinc sulfide (ZnS), aluminum, aluminum oxide,stainless steel, Kapton™, metal, ceramic, plastic, polymer, sapphire,silicon carbide, silicon on insulator (SOI), astrosital, barium borate,barium fluoride, sillenite crystals BGO/BSO/BTO, bismuth germanate,calcite, calcium fluoride, cesium iodide, FeILiNbO₃, fused quartz,quartz, fused silica, glass, SiO₂, gallium, gadolinium garnet, potassiumdihydrogen phosphate (KDP), KRS-5, potassium titanyl phosphate, leadmolibdate, lithium fluoride, lithium iodate, lithium niobate, lithiumtantalate, magnesium fluoride, potassium bromide, titanium dioixde,sodium chloride, tellurium dioxide, zinc selenide, spin-on glass, UVcurable materials, soda lime glass, any compound above in anhydrogenated form, stoichiometric variations thereof, or any combinationthereof.
 105. The method of claim 101, wherein the first substratecomprises a thickness of about 10 nm to about 10,000 nm.
 106. The methodof claim 101, wherein the first front-end branched channel regioncomprises a splitter structure dividing the primary channel into leasttwo secondary channels.
 107. The method of claim 106, wherein thesplitter structure comprises at least one surface angled in the range offrom about 0 and about 90 degrees relative to the centerline of theprimary channel.
 108. The method of claim 101, wherein the width of eachsecondary channel is about 30% to about 70% of the width of the primarychannel.
 109. The method of claim 101, wherein the length of a secondarychannel is about 1 micron to about 500 microns.
 110. The method of claim106, wherein each of the secondary channel is divided into two tertiarychannels by a splitter comprising at least one surface angled in therange of from about 0 and about 90 degrees relative to the centerline ofthe secondary channel.
 111. The method of claim 106, wherein thesplitter is configured so as to define an overhang that shields at leasta portion of the secondary channel from the primary channel.
 112. Themethod of claim 101, wherein at least one of the plurality ofnanochannels comprises a varying width, a varying depth, or both. 113.The method of claim 101, wherein the length of each of the plurality ofnanochannels is from about 50 microns to about 5000 microns.
 114. Themethod of claim 106, wherein the splitter structure comprises acontoured portion.
 115. The method of claim 114, wherein the splitterstructure is configured such that a fluidborne body propelled throughthe primary channel by a gradient is essentially equally likely to entereither secondary channel downstream from the splitter structure. 116.The method of claim 111, wherein the overhang is about 5% to about 50%of the width of the secondary channel.
 117. The method of claim 101,further comprising disposing a thin film atop at least a portion of thefirst substrate, the second substrate, or both, the film enhancingbonding between the substrates.
 118. The method of claim 117, whereinthe thin film electrically insulates at least a portion of the interiorof the enclosed conduit from at least one of the substrates.
 119. Themethod of claim 117, wherein the thin film comprises a material selectedfrom the group consisting of silicon nitride, silicon oxynitride,SiOxNy, hydrogenated silicon dioxide, hydrogenated silicon nitride,hydrogenated silicon oxinitride, high K dielectrics, TiSiO, TiO, TiN,titanium oxides, hydrogenatedtitanium oxides, titanium nitrides,hydrogenated titanium nitrides, TaO, TaSiO, TaOxNy, Ta₂O₅, TaCN,tantalum oxides, hydrogenated tantalum oxides, tantalum nitrides,hydrogenated tantalum nitrides, HfO₂, HfSiO₂, HfZrO_(x), HfN, HfON,HfSiN, HfSiON, hafnium oxides, hydrogenated hafnium oxides, hafniumnitrides, hydrogenated hafnium nitrides, ZrO₂, ZrSiO₂, ZrN, ZrSiN, ZrON,ZrSiON, zirconium oxides, hydrogenated zirconium oxides, zirconiumnitrides, hydrogenated zirconium nitrides, Al₂O₃, AlN, TiAlN, TaAlN,WAlN, aluminum oxides, hydrogenated aluminum oxides, aluminum nitrides,hydrogenated aluminum nitrides, SiN, WN, low K dielectrics, fluorinedoped silicon dioxide, carbon doped silicon dioxide, porous silicondioxide, porous carbon doped silicon dioxide, spin-on organic polymericdielectrics, graphite, graphene, carbon nano-tubes, plastics, polymer,organic molecules, self-assembled monolayers, self-assembledmulti-layers, a lipid bi-layer, any of the aforementioned compounds in ahydrogenated form, a stoichiometric variation of any of the foregoing,and any combination thereof.
 120. A method of analysis, comprising:translocating a DNA molecule through at least two channels ofsuccessively decreasing width such that at least a portion of themacromolecule is elongated while disposed in the narrowest of thechannels, wherein the widest channel has a width in the range of fromabout 10 nm to less than about 10000 nm, wherein the ratio of the widthsof the widest and narrowest channels is in the range of from about 100to about 10000; detecting a signal from the DNA molecule while the DNAmolecule resides in a region of the narrowest channel; and correlatingthe signal to a property of the DNA molecule.